Dna polymerases with increased  3&#39;-mismatch discrimination

ABSTRACT

Disclosed are mutant DNA polymerases having increased 3′-mismatch discrimination relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present invention claims benefit of priority to U.S. ProvisionalPatent Application No. 61/356,280, filed Jun. 18, 2010, which isincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides DNA polymerases with increased3′-mismatch discrimination and their use in various applications,including nucleic acid polynucleotide extension and amplification.

BACKGROUND OF THE INVENTION

DNA polymerases are responsible for the replication and maintenance ofthe genome, a role that is central to accurately transmitting geneticinformation from generation to generation. DNA polymerases function incells as the enzymes responsible for the synthesis of DNA. Theypolymerize deoxyribonucleoside triphosphates in the presence of a metalactivator, such as Mg²⁺, in an order dictated by the DNA template orpolynucleotide template that is copied. In vivo, DNA polymerasesparticipate in a spectrum of DNA synthetic processes including DNAreplication, DNA repair, recombination, and gene amplification. Duringeach DNA synthetic process, the DNA template is copied once or at most afew times to produce identical replicas. In contrast, in vitro, DNAreplication can be repeated many times such as, for example, duringpolymerase chain reaction (see, e.g., U.S. Pat. No. 4,683,202).

In the initial studies with polymerase chain reaction (PCR), the DNApolymerase was added at the start of each round of DNA replication (seeU.S. Pat. No. 4,683,202, supra). Subsequently, it was determined thatthermostable DNA polymerases could be obtained from bacteria that growat elevated temperatures, and that these enzymes need to be added onlyonce (see U.S. Pat. No. 4,889,818 to Gelfand and U.S. Pat. No. 4,965,188to Mullis). At the elevated temperatures used during PCR, these enzymesare not irreversibly inactivated. As a result, one can carry outrepetitive cycles of polymerase chain reactions without adding freshenzymes at the start of each synthetic addition process. DNApolymerases, particularly thermostable polymerases, are the key to alarge number of techniques in recombinant DNA studies and in medicaldiagnosis of disease. For diagnostic applications in particular, atarget nucleic acid sequence may be only a small portion of the DNA orRNA in question, so it may be difficult to detect the presence of atarget nucleic acid sequence without amplification.

The overall folding pattern of DNA polymerases resembles the human righthand and contains three distinct subdomains of palm, fingers, and thumb.(See Beese et al., Science 260:352-355, 1993); Patel et al.,Biochemistry 34:5351-5363, 1995). While the structure of the fingers andthumb subdomains vary greatly between polymerases that differ in sizeand in cellular functions, the catalytic palm subdomains are allsuperimposable. For example, motif A, which interacts with the incomingdNTP and stabilizes the transition state during chemical catalysis, issuperimposable with a mean deviation of about one Å amongst mammalianpol α and prokaryotic pol I family DNA polymerases (Wang et al., Cell89:1087-1099, 1997). Motif A begins structurally at an antiparallelβ-strand containing predominantly hydrophobic residues and continues toan α-helix. The primary amino acid sequence of DNA polymerase activesites is exceptionally conserved. In the case of motif A, for example,the sequence DYSQIELR (SEQ ID NO:28) is retained in polymerases fromorganisms separated by many millions years of evolution, including,e.g., Thermus aquaticus, Chlamydia trachomatis, and Escherichia coli.

In addition to being well-conserved, the active site of DNA polymeraseshas also been shown to be relatively mutable, capable of accommodatingcertain amino acid substitutions without reducing DNA polymeraseactivity significantly. (See, e.g., U.S. Pat. No. 6,602,695) Such mutantDNA polymerases can offer various selective advantages in, e.g.,diagnostic and research applications comprising nucleic acid synthesisreactions. Thus, there is a need in the art for identification of aminoacid positions amenable to mutation to yield improved polymeraseactivities. The present invention, as set forth herein, meets these andother needs.

BRIEF SUMMARY OF THE INVENTION

Provided herein are DNA polymerases having increased 3′-mismatchdiscrimination relative to a corresponding, unmodified controlpolymerase, and methods of making and using such DNA polymerases. Insome embodiments, the polymerase is a thermostable DNA polymerase. Insome embodiments, the DNA polymerase is a thermoactive DNA polymerase.

In some embodiments, the DNA polymerase is derived from a Thermusspecies. In some embodiments, the DNA polymerase is derived from aThermotoga species. In some embodiments, the amino acid of the DNApolymerase corresponding to position 547 of SEQ ID NO:1 is any aminoacid other than Y, and the control DNA polymerase has the same aminoacid sequence as the DNA polymerase except that the amino acid of thecontrol DNA polymerase corresponding to position 547 of SEQ ID NO:1 isY. For example, in some embodiments, the amino acid at the positioncorresponding to position 547 of SEQ ID NO:1 is selected from G, A, V,L, I, M, F, W, P, S, T, C, N, Q, D, E, K, R or H. In some embodiments,the amino acid at the position corresponding to position 547 of SEQ IDNO:1 is an amino acid having a non-polar, uncharged side-chain (e.g., G,A, L, M, W, P, F, C, V, or I) or a polar, uncharged side-chain (e.g., N,Q, H, S, or T). In some embodiments, the amino acid at the positioncorresponding to position 547 of SEQ ID NO:1 is C or N. In someembodiments, the amino acid at the position corresponding to position547 of SEQ ID NO:1 is C. In some embodiments, the amino acid at theposition corresponding to position 547 of SEQ ID NO:1 is N.

In some embodiments, the DNA polymerase having increased 3′-mismatchdiscrimination comprises a motif in the polymerase domain comprising

-   -   K-L-X₁-X₂-T-X₃-X₄-X₅-X₆-X₇-X₈, wherein:    -   X₁ is K, R or Q;    -   X₂ is N, S, or G;    -   X₃ is any amino acid other than Y;    -   X₄ is V, I or L;    -   X₅ is D or E;    -   X₆ is P, A, T, S or G;    -   X₇ is L or I; and    -   X₈ is P or L (SEQ ID NO:8).

In some embodiments, the DNA polymerase having increased 3′-mismatchdiscrimination comprises a motif in the polymerase domain comprising

K-L-K-X₂-T-X₃-X₄-D-X₆-L-P, wherein:

-   -   X₂ is N or S;    -   X₃ is any amino acid other than Y;    -   X₄ is V or I; and    -   X₆ is P or A (SEQ ID NO:9).

In some embodiments, the DNA polymerase having increased 3′-mismatchdiscrimination comprises a motif in the polymerase domain comprisingK-L-K-X₂-T-X₃-X₄-D-P-L-P, wherein:

-   -   X₂ is N or S;    -   X₃ is any amino acid other than Y; and    -   X₄ is V or I (SEQ ID NO:10).

In some embodiments, X₃ is an amino acid having a nonpolar, unchargedside chain (i.e., C, I, L, M, F, P, W, or V) or a polar uncharged sidechain (other than Y, i.e., N, Q, H, S, or T).

In some embodiments, X₃ is C or N (SEQ ID NO:11).

In some embodiments, the amino acid of the DNA polymerase correspondingto position 580 of SEQ ID NO:1 is any amino acid other than D or E. Insome embodiments, the amino acid of the DNA polymerase corresponding toposition 580 of SEQ ID NO:1 is any amino acid other than D. In someembodiments, the amino acid of the DNA polymerase corresponding toposition 580 of SEQ ID NO:1 is selected from the group consisting of L,G, T, Q, A, S, N, R and K. In some embodiments, the amino acid of theDNA polymerase corresponding to position 580 of SEQ ID NO:1 is G.

Various DNA polymerases are amenable to mutation according to thepresent invention. Particularly suitable are thermostable polymerases,including wild-type or naturally occurring thermostable polymerases fromvarious species of thermophilic bacteria, as well as syntheticthermostable polymerases derived from such wild-type or naturallyoccurring enzymes by amino acid substitution, insertion, or deletion, orother modification. Exemplary unmodified forms of polymerase include,e.g., CS5 (SEQ ID NO:29), CS6 (SEQ ID NO:30) or Z05 DNA polymerase (SEQID NO:1), or a functional DNA polymerase having at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity thereto. Other unmodified polymerasesinclude, e.g., DNA polymerases from any of the following species ofthermophilic bacteria (or a functional DNA polymerase having at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity to such a polymerase):Thermotoga maritima (SEQ ID NO:38); Thermus aquaticus (SEQ ID NO:2);Thermus thermophilus (SEQ ID NO:6); Thermus flavus (SEQ ID NO:4);Thermus filiformis (SEQ ID NO:3); Thermus sp. Sps17 (SEQ ID NO:5);Thermus sp. Z05 (SEQ ID NO:1); Thermotoga neopolitana (SEQ ID NO:39);Thermosipho africanus (SEQ ID NO:37); Thermus caldophilus (SEQ ID NO:7),Deinococcus radiodurans (SEQ ID NO:36), Bacillus stearothermophilus (SEQID NO:40) or Bacillus caldotenax (SEQ ID NO:41). Suitable polymerasesalso include those having reverse transcriptase (RT) activity and/or theability to incorporate unconventional nucleotides, such asribonucleotides or other 2′-modified nucleotides.

While thermostable DNA polymerases possessing efficient 3′-mismatchdiscrimination activity are particularly suited for performing PCR,thermoactive, but not thermostable DNA polymerases possessing efficient3′-mismatch discrimination activity also are amenable to mutationaccording to the present invention.

In some embodiments, the DNA polymerase is a Thermus DNA polymerase. Forexample, in some embodiments, the DNA polymerase has at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity to a polymerase selected from thegroup consisting of:

-   -   (a) a Thermus sp. Z05 DNA polymerase (Z05) (SEQ ID NO:1);    -   (b) a Thermus aquaticus DNA polymerase (Taq) (SEQ ID NO:2);    -   (c) a Thermus filiformis DNA polymerase (Tfi) (SEQ ID NO:3);    -   (d) a Thermus flavus DNA polymerase (Tfl) (SEQ ID NO:4);    -   (e) a Thermus sp. Sps17 DNA polymerase (Sps17) (SEQ ID NO:5);    -   (f) a Thermus thermophilus DNA polymerase (Tth) (SEQ ID NO:6);        and    -   (g) aThermus caldophilus DNA polymerase (Tca) (SEQ ID NO:7).

In some embodiments, the DNA polymerase is a Thermotoga DNA polymerase.For example, in some embodiments, the DNA polymerase has at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% sequence identity to a polymerase selected fromthe group consisting of:

-   -   (a) a Thermotoga maritima DNA polymerase (Tma) (SEQ ID NO:38);    -   (b) a Thermotoga neopolitana DNA polymerase (Tne) (SEQ ID        NO:39);

In some embodiments, the DNA polymerase has at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% sequence identity to SEQ ID NO:1. In some embodiments, the DNApolymerase is a Thermus sp. Z05 DNA polymerase (Z05) DNA polymerase(i.e., SEQ ID NO:1), except that the amino acid at position 547 is anyamino acid other than Y. For example, in some embodiments, the aminoacid at position 547 is selected from G, A, V, L, I, M, F, W, P, S, T,C, N, Q, D, E, K, R or H. In some embodiments, the DNA polymerase is aZ05 DNA polymerase, and the amino acid at position 547 is any amino acidother than Y. In some embodiments, the DNA polymerase is a Z05 DNApolymerase, and the amino acid at position 547 is C or N. In someembodiments, the DNA polymerase is a Z05 DNA polymerase furthercomprising a substitution at position 580, and the amino acid atposition 580 is any amino acid other than D or E. In some embodiments,the DNA polymerase is a Z05 DNA polymerase, and the amino acid atposition 580 is any amino acid other than D. In some embodiments, theDNA polymerase is a Z05 DNA polymerase, and the amino acid at position580 is selected from the group consisting of L, G, T, Q, A, S, N, R andK. In some embodiments, the DNA polymerase is a Z05 DNA polymerase, andthe amino acid at position 580 is G.

The mutant or improved polymerase can include other, non-substitutionalmodifications. One such modification is a thermally reversible covalentmodification that inactivates the enzyme, but which is reversed toactivate the enzyme upon incubation at an elevated temperature, such asa temperature typically used for polynucleotide extension. Exemplaryreagents for such thermally reversible modifications are described inU.S. Pat. Nos. 5,773,258 and 5,677,152 to Birch et al., which areexpressly incorporated by reference herein in their entirety.

In some embodiments, the 3′-mismatch activity is determined using amutant BRAF V600R target polynucleotide having the nucleic acid sequenceof SEQ ID NO:35 (wild type BRAF=SEQ ID NO:34) in the presence of aforward primer that is perfectly matched to the mutant sequence and hasa single 3′ A:C mismatch to the wild type sequence in one or morereaction mixtures having a predetermined number of copies of wild-typeBRAF V600 target polynucleotide and a predetermined number of copies ofmutant BRAF V600R target polynucleotide equal in number or fewer thanthe number of copies of wild-type target (e.g., 10,000 or fewer copies).Two or more reaction mixtures can have titrated numbers of copies ofmutant BRAF V600R target polynucleotide (e.g., 1:5 titrations, 1:10titrations, e.g., 10,000 copies, 1000 copies, 100 copies, 10 copies, 1copy, 0 copies in several reaction mixtures). The 3′-mismatchdiscrimination ability of a polymerase of the invention can be comparedto the 3′-mismatch discrimination ability of a reference polymerase(e.g., a naturally occurring or unmodified polymerase), over apreselected unit of time, as described herein. Polymerases withincreased 3′-mismatch discrimination ability will not amplify thewild-type sequence when contacted with a primer that is perfectlymatched to a mutant allele, or will require a greater number of PCRcycles to amplify the wild-type sequence using the mutantallele-specific primer (i.e., exhibit a higher Cp value), in comparisonto a naturally occurring or unmodified polymerase.

In various other aspects, the present invention provides a recombinantnucleic acid encoding a mutant or improved DNA polymerase as describedherein, a vector comprising the recombinant nucleic acid, and/or a hostcell transformed with the vector. In certain embodiments, the vector isan expression vector. Host cells comprising such expression vectors areuseful in methods of the invention for producing the mutant or improvedpolymerase by culturing the host cells under conditions suitable forexpression of the recombinant nucleic acid. The polymerases of theinvention may be contained in reaction mixtures and/or kits. Theembodiments of the recombinant nucleic acids, host cells, vectors,expression vectors, reaction mixtures and kits are as described aboveand herein.

In yet another aspect, a method for conducting polynucleotide extensionis provided. The method generally includes contacting a DNA polymerasehaving increased 3′-mismatch discrimination as described herein with aprimer, a polynucleotide template, and nucleoside triphosphates underconditions suitable for extension of the primer, thereby producing anextended primer. The polynucleotide template can be, for example, an RNAor DNA template. The nucleoside triphosphates can include unconventionalnucleotides such as, e.g., ribonucleotides and/or labeled nucleotides.Further, the primer and/or template can include one or more nucleotideanalogs. In some variations, the polynucleotide extension method is amethod for polynucleotide amplification that includes contacting themutant or improved DNA polymerase with a primer pair, the polynucleotidetemplate, and the nucleoside triphosphates under conditions suitable foramplification of the polynucleotide. The polynucleotide extensionreaction can be, e.g., PCR, isothermal extension, or sequencing (e.g.,454 sequencing reaction).

The present invention also provides a kit useful in such apolynucleotide extension method. Generally, the kit includes at leastone container providing a mutant or improved DNA polymerase as describedherein. In certain embodiments, the kit further includes one or moreadditional containers providing one or more additional reagents. Forexample, in specific variations, the one or more additional containersprovide nucleoside triphosphates; a buffer suitable for polynucleotideextension; and/or a primer hybridizable, under polynucleotide extensionconditions, to a predetermined polynucleotide template.

Further provided are reaction mixtures comprising the polymerases of theinvention. The reactions mixtures can also contain a template nucleicacid (DNA and/or RNA), one or more primer or probe polynucleotides,nucleoside triphosphates (including, e.g., deoxyribonucleotides,ribonucleotides, labeled nucleotides, unconventional nucleotides),buffers, salts, labels (e.g., fluorophores).

Further embodiments of the invention are described herein.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although essentially anymethods and materials similar to those described herein can be used inthe practice or testing of the present invention, only exemplary methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

The terms “a,” “an,” and “the” include plural referents, unless thecontext clearly indicates otherwise.

An “amino acid” refers to any monomer unit that can be incorporated intoa peptide, polypeptide, or protein. As used herein, the term “aminoacid” includes the following twenty natural or genetically encodedalpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine(Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine(Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K),methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P),serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine(Tyr or Y), and valine (Val or V). In cases where “X” residues areundefined, these should be defined as “any amino acid.” The structuresof these twenty natural amino acids are shown in, e.g., Stryer et al.,Biochemistry, 5^(th) ed., Freeman and Company (2002), which isincorporated by reference. Additional amino acids, such asselenocysteine and pyrrolysine, can also be genetically coded for(Stadtman (1996) “Selenocysteine,” Annu Rev Biochem. 65:83-100 and Ibbaet al. (2002) “Genetic code: introducing pyrrolysine,” Curr Biol.12(13):R464-R466, which are both incorporated by reference). The term“amino acid” also includes unnatural amino acids, modified amino acids(e.g., having modified side chains and/or backbones), and amino acidanalogs. See, e.g., Zhang et al. (2004) “Selective incorporation of5-hydroxytryptophan into proteins in mammalian cells,” Proc. Natl. Acad.Sci. U.S.A. 101(24):8882-8887, Anderson et al.

(2004) “An expanded genetic code with a functional quadruplet codon”Proc. Natl. Acad. Sci. U.S.A. 101(20):7566-7571, Ikeda et al. (2003)“Synthesis of a novel histidine analogue and its efficient incorporationinto a protein in vivo,” Protein Eng. Des. Sel. 16(9):699-706, Chin etal. (2003) “An Expanded Eukaryotic Genetic Code,” Science301(5635):964-967, James et al. (2001) “Kinetic characterization ofribonuclease S mutants containing photoisomerizablephenylazophenylalanine residues,” Protein Eng. Des. Sel. 14(12):983-991,Kohrer et al. (2001) “Import of amber and ochre suppressor tRNAs intomammalian cells: A general approach to site-specific insertion of aminoacid analogues into proteins,” Proc. Natl. Acad. Sci. U.S.A.98(25):14310-14315, Bacher et al. (2001) “Selection and Characterizationof Escherichia coli Variants Capable of Growth on an Otherwise ToxicTryptophan Analogue,” J. Bacteriol. 183(18):5414-5425, Hamano-Takaku etal. (2000) “A Mutant Escherichia coli Tyrosyl-tRNA Synthetase Utilizesthe Unnatural Amino Acid Azatyrosine More Efficiently than Tyrosine,” J.Biol. Chem. 275(51):40324-40328, and Budisa et al. (2001) “Proteins with{beta}-(thienopyrrolyl)alanines as alternative chromophores andpharmaceutically active amino acids,” Protein Sci. 10(7):1281-1292,which are each incorporated by reference.

To further illustrate, an amino acid is typically an organic acid thatincludes a substituted or unsubstituted amino group, a substituted orunsubstituted carboxy group, and one or more side chains or groups, oranalogs of any of these groups. Exemplary side chains include, e.g.,thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl,hydrazine, cyano, halo, hydrazide, alkenyl, alkynl, ether, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, ester, thioacid, hydroxylamine, or any combination of thesegroups. Other representative amino acids include, but are not limitedto, amino acids comprising photoactivatable cross-linkers, metal bindingamino acids, spin-labeled amino acids, fluorescent amino acids,metal-containing amino acids, amino acids with novel functional groups,amino acids that covalently or noncovalently interact with othermolecules, photocaged and/or photoisomerizable amino acids, radioactiveamino acids, amino acids comprising biotin or a biotin analog,glycosylated amino acids, other carbohydrate modified amino acids, aminoacids comprising polyethylene glycol or polyether, heavy atomsubstituted amino acids, chemically cleavable and/or photocleavableamino acids, carbon-linked sugar-containing amino acids, redox-activeamino acids, amino thioacid containing amino acids, and amino acidscomprising one or more toxic moieties.

The term “aptamer” refers to a single-stranded DNA that recognizes andbinds to DNA polymerase, and efficiently inhibits the polymeraseactivity as described in U.S. Pat. No. 5,693,502, hereby expresslyincorporated by reference herein in its entirety.

The term “mutant,” in the context of DNA polymerases of the presentinvention, means a polypeptide, typically recombinant, that comprisesone or more amino acid substitutions relative to a corresponding,naturally-occurring or unmodified DNA polymerase.

The term “unmodified form,” in the context of a mutant polymerase, is aterm used herein for purposes of defining a mutant DNA polymerase of thepresent invention: the term “unmodified form” refers to a functional DNApolymerase that has the amino acid sequence of the mutant polymeraseexcept at one or more amino acid position(s) specified as characterizingthe mutant polymerase. Thus, reference to a mutant DNA polymerase interms of (a) its unmodified form and (b) one or more specified aminoacid substitutions means that, with the exception of the specified aminoacid substitution(s), the mutant polymerase otherwise has an amino acidsequence identical to the unmodified form in the specified motif. The“unmodified polymerase” (and therefore also the modified form havingincreased 3′-mismatch discrimination) may contain additional mutationsto provide desired functionality, e.g., improved incorporation ofdideoxyribonucleotides, ribonucleotides, ribonucleotide analogs,dye-labeled nucleotides, modulating 5′-nuclease activity, modulating3′-nuclease (or proofreading) activity, or the like. Accordingly, incarrying out the present invention as described herein, the unmodifiedform of a DNA polymerase is predetermined. The unmodified form of a DNApolymerase can be, for example, a wild-type and/or a naturally occurringDNA polymerase, or a DNA polymerase that has already been intentionallymodified. An unmodified form of the polymerase is preferably athermostable DNA polymerases, such as DNA polymerases from variousthermophilic bacteria, as well as functional variants thereof havingsubstantial sequence identity to a wild-type or naturally occurringthermostable polymerase. Such variants can include, for example,chimeric DNA polymerases such as, for example, the chimeric DNApolymerases described in U.S. Pat. No. 6,228,628 and U.S. ApplicationPublication No. 2004/0005599, which are incorporated by reference hereinin their entirety. In certain embodiments, the unmodified form of apolymerase has reverse transcriptase (RT) activity.

The term “thermostable polymerase,” refers to an enzyme that is stableto heat, is heat resistant, and retains sufficient activity to effectsubsequent polynucleotide extension reactions and does not becomeirreversibly denatured (inactivated) when subjected to the elevatedtemperatures for the time necessary to effect denaturation ofdouble-stranded nucleic acids. The heating conditions necessary fornucleic acid denaturation are well known in the art and are exemplifiedin, e.g., U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,965,188, which areincorporated herein by reference. As used herein, a thermostablepolymerase is suitable for use in a temperature cycling reaction such asthe polymerase chain reaction (“PCR”). Irreversible denaturation forpurposes herein refers to permanent and complete loss of enzymaticactivity. For a thermostable polymerase, enzymatic activity refers tothe catalysis of the combination of the nucleotides in the proper mannerto form polynucleotide extension products that are complementary to atemplate nucleic acid strand. Thermostable DNA polymerases fromthermophilic bacteria include, e.g., DNA polymerases from Thermotogamaritima, Thermus aquaticus, Thermus thermophilus, Thermus flavus,Thermus filiformis, Thermus species Sps17, Thermus species Z05, Thermuscaldophilus, Bacillus caldotenax, Thermotoga neopolitana, andThermosipho africanus.

The term “thermoactive” refers to an enzyme that maintains catalyticproperties at temperatures commonly used for reverse transcription oranneal/extension steps in RT-PCR and/or PCR reactions (i.e., 45-80° C.).Thermostable enzymes are those which are not irreversibly inactivated ordenatured when subjected to elevated temperatures necessary for nucleicacid denaturation. Thermoactive enzymes may or may not be thermostable.Thermoactive DNA polymerases can be DNA or RNA dependent fromthermophilic species or from mesophilic species including, but notlimited to, Escherichia coli, Moloney murine leukemia viruses, and Avianmyoblastosis virus.

As used herein, a “chimeric” protein refers to a protein whose aminoacid sequence represents a fusion product of subsequences of the aminoacid sequences from at least two distinct proteins. A chimeric proteintypically is not produced by direct manipulation of amino acidsequences, but, rather, is expressed from a “chimeric” gene that encodesthe chimeric amino acid sequence. In certain embodiments, for example,an unmodified form of a mutant DNA polymerase of the present inventionis a chimeric protein that consists of an amino-terminal (N-terminal)region derived from a Thermus species DNA polymerase and acarboxy-terminal (C-terminal) region derived from Tma DNA polymerase.The N-terminal region refers to a region extending from the N-terminus(amino acid position 1) to an internal amino acid. Similarly, theC-terminal region refers to a region extending from an internal aminoacid to the C-terminus.

In the context of DNA polymerases, “correspondence” to another sequence(e.g., regions, fragments, nucleotide or amino acid positions, or thelike) is based on the convention of numbering according to nucleotide oramino acid position number and then aligning the sequences in a mannerthat maximizes the percentage of sequence identity. Because not allpositions within a given “corresponding region” need be identical,non-matching positions within a corresponding region may be regarded as“corresponding positions.” Accordingly, as used herein, referral to an“amino acid position corresponding to amino acid position [X]” of aspecified DNA polymerase refers to equivalent positions, based onalignment, in other DNA polymerases and structural homologues andfamilies. In some embodiments of the present invention, “correspondence”of amino acid positions are determined with respect to a region of thepolymerase comprising one or more motifs of SEQ ID NO:1, 2, 3, 4, 5, 6,7, 36, 37, 38, 39, 40, or 41. When a polymerase polypeptide sequencediffers from SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 36, 37, 38, 39, 40, or 41(e.g., by changes in amino acids or addition or deletion of aminoacids), it may be that a particular mutation associated with improvedactivity as discussed herein will not be in the same position number asit is in SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 36, 37, 38, 39, 40, or 41. Thisis illustrated, for example, in Table 1.

“Recombinant,” as used herein, refers to an amino acid sequence or anucleotide sequence that has been intentionally modified by recombinantmethods. By the term “recombinant nucleic acid” herein is meant anucleic acid, originally formed in vitro, in general, by themanipulation of a nucleic acid by endonucleases, in a form not normallyfound in nature. Thus an isolated, mutant DNA polymerase nucleic acid,in a linear form, or an expression vector foamed in vitro by ligatingDNA molecules that are not normally joined, are both consideredrecombinant for the purposes of this invention. It is understood thatonce a recombinant nucleic acid is made and reintroduced into a hostcell, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. A “recombinant protein”is a protein made using recombinant techniques, i.e., through theexpression of a recombinant nucleic acid as depicted above.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation.

The term “host cell” refers to both single-cellular prokaryote andeukaryote organisms (e.g., bacteria, yeast, and actinomycetes) andsingle cells from higher order plants or animals when being grown incell culture.

The term “vector” refers to a piece of DNA, typically double-stranded,which may have inserted into it a piece of foreign DNA. The vector ormay be, for example, of plasmid origin. Vectors contain “replicon”polynucleotide sequences that facilitate the autonomous replication ofthe vector in a host cell. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell, which, for example,replicates the vector molecule, encodes a selectable or screenablemarker, or encodes a transgene. The vector is used to transport theforeign or heterologous DNA into a suitable host cell. Once in the hostcell, the vector can replicate independently of or coincidental with thehost chromosomal DNA, and several copies of the vector and its insertedDNA can be generated. In addition, the vector can also contain thenecessary elements that permit transcription of the inserted DNA into anmRNA molecule or otherwise cause replication of the inserted DNA intomultiple copies of RNA. Some expression vectors additionally containsequence elements adjacent to the inserted DNA that increase thehalf-life of the expressed mRNA and/or allow translation of the mRNAinto a protein molecule. Many molecules of mRNA and polypeptide encodedby the inserted DNA can thus be rapidly synthesized.

The term “nucleotide,” in addition to referring to the naturallyoccurring ribonucleotide or deoxyribonucleotide monomers, shall hereinbe understood to refer to related structural variants thereof, includingderivatives and analogs, that are functionally equivalent with respectto the particular context in which the nucleotide is being used (e.g.,hybridization to a complementary base), unless the context clearlyindicates otherwise.

The term “nucleic acid” or “polynucleotide” refers to a polymer that canbe corresponded to a ribose nucleic acid (RNA) or deoxyribose nucleicacid (DNA) polymer, or an analog thereof. This includes polymers ofnucleotides such as RNA and DNA, as well as synthetic forms, modified(e.g., chemically or biochemically modified) forms thereof, and mixedpolymers (e.g., including both RNA and DNA subunits). Exemplarymodifications include methylation, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, and the like), pendentmoieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen,and the like), chelators, alkylators, and modified linkages (e.g., alphaanomeric nucleic acids and the like). Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions. Typically, the nucleotide monomers are linked viaphosphodiester bonds, although synthetic forms of nucleic acids cancomprise other linkages (e.g., peptide nucleic acids as described inNielsen et al. (Science 254:1497-1500, 1991). A nucleic acid can be orcan include, e.g., a chromosome or chromosomal segment, a vector (e.g.,an expression vector), an expression cassette, a naked DNA or RNApolymer, the product of a polymerase chain reaction (PCR), anoligonucleotide, a probe, and a primer. A nucleic acid can be, e.g.,single-stranded, double-stranded, or triple-stranded and is not limitedto any particular length. Unless otherwise indicated, a particularnucleic acid sequence comprises or encodes complementary sequences, inaddition to any sequence explicitly indicated.

The term “oligonucleotide” refers to a nucleic acid that includes atleast two nucleic acid monomer units (e.g., nucleotides). Anoligonucleotide typically includes from about six to about 175 nucleicacid monomer units, more typically from about eight to about 100 nucleicacid monomer units, and still more typically from about 10 to about 50nucleic acid monomer units (e.g., about 15, about 20, about 25, about30, about 35, or more nucleic acid monomer units). The exact size of anoligonucleotide will depend on many factors, including the ultimatefunction or use of the oligonucleotide. Oligonucleotides are optionallyprepared by any suitable method, including, but not limited to,isolation of an existing or natural sequence, DNA replication oramplification, reverse transcription, cloning and restriction digestionof appropriate sequences, or direct chemical synthesis by a method suchas the phosphotriester method of Narang et al. (Meth. Enzymol. 68:90-99,1979); the phosphodiester method of Brown et al. (Meth. Enzymol.68:109-151, 1979); the diethylphosphoramidite method of Beaucage et al.(Tetrahedron Lett. 22:1859-1862, 1981); the triester method of Matteucciet al. (J. Am. Chem. Soc. 103:3185-3191, 1981); automated synthesismethods; or the solid support method of U.S. Pat. No. 4,458,066,entitled “PROCESS FOR PREPARING POLYNUCLEOTIDES,” issued Jul. 3, 1984 toCaruthers et al., or other methods known to those skilled in the art.All of these references are incorporated by reference.

The term “primer” as used herein refers to a polynucleotide capable ofacting as a point of initiation of template-directed nucleic acidsynthesis when placed under conditions in which polynucleotide extensionis initiated (e.g., under conditions comprising the presence ofrequisite nucleoside triphosphates (as dictated by the template that iscopied) and a polymerase in an appropriate buffer and at a suitabletemperature or cycle(s) of temperatures (e.g., as in a polymerase chainreaction)). To further illustrate, primers can also be used in a varietyof other oligonuceotide-mediated synthesis processes, including asinitiators of de novo RNA synthesis and in vitro transcription-relatedprocesses (e.g., nucleic acid sequence-based amplification (NASBA),transcription mediated amplification (TMA), etc.). A primer is typicallya single-stranded oligonucleotide (e.g., oligodeoxyribonucleotide). Theappropriate length of a primer depends on the intended use of the primerbut typically ranges from 6 to 40 nucleotides, more typically from 15 to35 nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the templatebut must be sufficiently complementary to hybridize with a template forprimer elongation to occur. In certain embodiments, the term “primerpair” means a set of primers including a 5′ sense primer (sometimescalled “forward”) that hybridizes with the complement of the 5′ end ofthe nucleic acid sequence to be amplified and a 3′ antisense primer(sometimes called “reverse”) that hybridizes with the 3′ end of thesequence to be amplified (e.g., if the target sequence is expressed asRNA or is an RNA). A primer can be labeled, if desired, by incorporatinga label detectable by spectroscopic, photochemical, biochemical,immunochemical, or chemical means. For example, useful labels include³²P, fluorescent dyes, electron-dense reagents, enzymes (as commonlyused in ELISA assays), biotin, or haptens and proteins for whichantisera or monoclonal antibodies are available.

The term “5′-nuclease probe” refers to an oligonucleotide that comprisesat least one light emitting labeling moiety and that is used in a5′-nuclease reaction to effect target nucleic acid detection. In someembodiments, for example, a 5′-nuclease probe includes only a singlelight emitting moiety (e.g., a fluorescent dye, etc.). In certainembodiments, 5′-nuclease probes include regions of self-complementaritysuch that the probes are capable of forming hairpin structures underselected conditions. To further illustrate, in some embodiments a5′-nuclease probe comprises at least two labeling moieties and emitsradiation of increased intensity after one of the two labels is cleavedor otherwise separated from the oligonucleotide. In certain embodiments,a 5′-nuclease probe is labeled with two different fluorescent dyes,e.g., a 5′ terminus reporter dye and the 3′ terminus quencher dye ormoiety. In some embodiments, 5′-nuclease probes are labeled at one ormore positions other than, or in addition to, terminal positions. Whenthe probe is intact, energy transfer typically occurs between the twofluorophores such that fluorescent emission from the reporter dye isquenched at least in part. During an extension step of a polymerasechain reaction, for example, a 5′-nuclease probe bound to a templatenucleic acid is cleaved by the 5′ to 3′ nuclease activity of, e.g., aTaq polymerase or another polymerase having this activity such that thefluorescent emission of the reporter dye is no longer quenched.Exemplary 5′-nuclease probes are also described in, e.g., U.S. Pat. No.5,210,015, entitled “Homogeneous assay system using the nucleaseactivity of a nucleic acid polymerase,” issued May 11, 1993 to Gelfandet al., U.S. Pat. No. 5,994,056, entitled “Homogeneous methods fornucleic acid amplification and detection,” issued Nov. 30, 1999 toHiguchi, and U.S. Pat. No. 6,171,785, entitled “Methods and devices forhomogeneous nucleic acid amplification and detector,” issued Jan. 9,2001 to Higuchi, which are each incorporated by reference herein. Inother embodiments, a 5′ nuclease probe may be labeled with two or moredifferent reporter dyes and a 3′ terminus quencher dye or moiety.

The term “FRET” or “fluorescent resonance energy transfer” or “Foersterresonance energy transfer” refers to a transfer of energy between atleast two chromophores, a donor chromophore and an acceptor chromophore(referred to as a quencher). The donor typically transfers the energy tothe acceptor when the donor is excited by light radiation with asuitable wavelength. The acceptor typically re-emits the transferredenergy in the form of light radiation with a different wavelength. Whenthe acceptor is a “dark” quencher, it dissipates the transferred energyin a form other than light. Whether a particular fluorophore acts as adonor or an acceptor depends on the properties of the other member ofthe FRET pair. Commonly used donor-acceptor pairs include the FAM-TAMRApair. Commonly used quenchers are DABCYL and TAMRA. Commonly used darkquenchers include BlackHole Quenchers™ (BHQ), (Biosearch Technologies,Inc., Novato, Cal.), Iowa Black™ (Integrated DNA Tech., Inc.,Coralville, Iowa), and BlackBerry™ Quencher 650 (BBQ-650) (Berry &Assoc., Dexter, Mich.).

The term “conventional” or “natural” when referring to nucleic acidbases, nucleoside triphosphates, or nucleotides refers to those whichoccur naturally in the polynucleotide being described (i.e., for DNAthese are dATP, dGTP, dCTP and dTTP). Additionally, dITP, and7-deaza-dGTP are frequently utilized in place of dGTP and 7-deaza-dATPcan be utilized in place of dATP in in vitro DNA synthesis reactions,such as sequencing. Collectively, these may be referred to as dNTPs.

The term “unconventional” or “modified” when referring to a nucleic acidbase, nucleoside, or nucleotide includes modification, derivations, oranalogues of conventional bases, nucleosides, or nucleotides thatnaturally occur in a particular polynucleotide. Certain unconventionalnucleotides are modified at the 2′ position of the ribose sugar incomparison to conventional dNTPs. Thus, although for RNA the naturallyoccurring nucleotides are ribonucleotides (i.e., ATP, GTP, CTP, UTP,collectively rNTPs), because these nucleotides have a hydroxyl group atthe 2′ position of the sugar, which, by comparison is absent in dNTPs,as used herein, ribonucleotides are unconventional nucleotides assubstrates for DNA polymerases. As used herein, unconventionalnucleotides include, but are not limited to, compounds used asterminators for nucleic acid sequencing. Exemplary terminator compoundsinclude but are not limited to those compounds that have a 2′,3′ dideoxystructure and are referred to as dideoxynucleoside triphosphates. Thedideoxynucleoside triphosphates ddATP, ddTTP, ddCTP and ddGTP arereferred to collectively as ddNTPs. Additional examples of terminatorcompounds include 2′-PO₄ analogs of ribonucleotides (see, e.g., U.S.Application Publication Nos. 2005/0037991 and 2005/0037398, which areboth incorporated by reference). Other unconventional nucleotidesinclude phosphorothioate dNTPs ([[α]-S]dNTPs), 5′-[α]-borano-dNTPs,[α]-methyl-phosphonate dNTPs, and ribonucleoside triphosphates (rNTPs).Unconventional bases may be labeled with radioactive isotopes such as³²P, ³³P, or ³⁵S; fluorescent labels; chemiluminescent labels;bioluminescent labels; hapten labels such as biotin; or enzyme labelssuch as streptavidin or avidin. Fluorescent labels may include dyes thatare negatively charged, such as dyes of the fluorescein family, or dyesthat are neutral in charge, such as dyes of the rhodamine family, ordyes that are positively charged, such as dyes of the cyanine family.Dyes of the fluorescein family include, e.g., FAM, HEX, TET, JOE, NANand ZOE. Dyes of the rhodamine family include Texas Red, ROX, R110, R6G,and TAMRA. Various dyes or nucleotides labeled with FAM, HEX, TET, JOE,NAN, ZOE, ROX, R110, R6G, Texas Red and TAMRA are marketed byPerkin-Elmer (Boston, Mass.), Applied Biosystems (Foster City, Calif.),or Invitrogen/Molecular Probes (Eugene, Oreg.). Dyes of the cyaninefamily include Cy2, Cy3, Cy5, and Cy7 and are marketed by GE HealthcareUK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).

As used herein, “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the sequence in the comparison window cancomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The terms “identical” or “identity,” in the context of two or morenucleic acids or polypeptide sequences, refer to two or more sequencesor subsequences that are the same. Sequences are “substantiallyidentical” to each other if they have a specified percentage ofnucleotides or amino acid residues that are the same (e.g., at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% identity over a specified region), when compared and aligned formaximum correspondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. These definitions also refer tothe complement of a test sequence. Optionally, the identity exists overa region that is at least about 50 nucleotides in length, or moretypically over a region that is 100 to 500 or 1000 or more nucleotidesin length.

The terms “similarity” or “percent similarity,” in the context of two ormore polypeptide sequences, refer to two or more sequences orsubsequences that have a specified percentage of amino acid residuesthat are either the same or similar as defined by a conservative aminoacid substitutions (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%,85%, 90%, or 95% similar over a specified region), when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection.Sequences are “substantially similar” to each other if they are at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, or at least 55% similar to each other. Optionally,this similarly exists over a region that is at least about 50 aminoacids in length, or more typically over a region that is at least about100 to 500 or 1000 or more amino acids in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters are commonly used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities or similarities for the test sequencesrelative to the reference sequence, based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, for example, by the local homologyalgorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by thehomology alignment algorithm of Needleman and Wunsch (J. Mol. Biol.48:443, 1970), by the search for similarity method of Pearson and Lipman(Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerizedimplementations of these algorithms (e.g., GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

Algorithms suitable for determining percent sequence identity andsequence similarity are the BLAST and BLAST 2.0 algorithms, which aredescribed in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977), andAltschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-87, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, typically less thanabout 0.01, and more typically less than about 0.001.

The term “mismatch discrimination” refers to the ability of abiocatalyst (e.g., an enzyme, such as a polymerase, ligase, or the like)to distinguish a fully complementary sequence from a mismatch-containingsequence when extending a nucleic acid (e.g., a primer or otheroligonucleotide) in a template-dependent manner by attaching (e.g.,covalently) one or more nucleotides to the nucleic acid. The term“3′-mismatch discrimination” refers to the ability of a biocatalyst todistinguish a fully complementary sequence from a mismatch-containing(nearly complementary) sequence where the nucleic acid to be extended(e.g., a primer or other oligonucleotide) has a mismatch at the nucleicacid's 3′ terminus compared to the template to which the nucleic acidhybridizes. In some embodiments, the nucleic acid to be extendedcomprises a mismatch at the 3′ end relative to the fully complementarysequence. In some embodiments, the nucleic acid to be extended comprisesa mismatch at the penultimate (N-1) 3′ position and/or at the N-2position relative to the fully complementary sequence.

The term “Cp value” or “crossing point” value refers to a value thatallows quantification of input target nucleic acids. The Cp value can bedetermined according to the second-derivative maximum method (VanLuu-The, et al., “Improved real-time RT-PCR method for high-throughputmeasurements using second derivative calculation and double correction,”BioTechniques, Vol. 38, No. 2, February 2005, pp. 287-293). In thesecond derivative method, a Cp corresponds to the first peak of a secondderivative curve. This peak corresponds to the beginning of a log-linearphase. The second derivative method calculates a second derivative valueof the real-time fluorescence intensity curve, and only one value isobtained. The original Cp method is based on a locally defined,differentiable approximation of the intensity values, e.g., by apolynomial function. Then the third derivative is computed. The Cp valueis the smallest root of the third derivative. The Cp can also bedetermined using the fit point method, in which the Cp is determined bythe intersection of a parallel to the threshold line in the log-linearregion (Van Luu-The, et al., BioTechniques, Vol. 38, No. 2, February2005, pp. 287-293). These computations are easily carried out by anyperson skilled in the art.

The term “PCR efficiency” refers to an indication of cycle to cycleamplification efficiency for the perfectly matched primer template. PCRefficiency is calculated for each condition using the equation: % PCRefficiency=(10^((−slope))−1)×100, wherein the slope was calculated bylinear regression with the log copy number plotted on the y-axis and Cpplotted on the x-axis.

The term “multiplex” refers to amplification with more than one set ofprimers, or the amplification of more that one polymorphism site in asingle reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an amino acid sequence alignment of a region from thepolymerase domain of exemplary DNA polymerases from various species ofbacteria: Thermus species Z05 (Z05) (SEQ ID NO:12), Thermus aquaticus(Taq) (SEQ ID NO:13), Thermus filiformus (Tfi) (SEQ ID NO:14), Thermusflavus (Tfl) (SEQ ID NO:15), Thermus species Sps17 (Sps17) (SEQ IDNO:16), Thermus thermophilus (Tth) (SEQ ID NO:17), Thermus caldophilus(Tca) (SEQ ID NO:18), Thermotoga maritima (Tma) (SEQ ID NO:19),Thermotoga neopolitana (Tne) (SEQ ID NO:20), Thermosipho africanus (Taf)(SEQ ID NO:21), Deinococcus radiodurans (Dra) (SEQ ID NO:23), Bacillusstearothermophilus (Bst) (SEQ ID NO:24), and Bacillus caldotenax (Bca)(SEQ ID NO:25). In addition, the polypeptide regions shown comprise theamino acid motif K-L-X₁-X₂-T-X₃-X₄-X₅-X₆-X₇-X₈ (SEQ ID NO:26), thevariable positions of which are further defined herein. This motif ishighlighted in bold type for each polymerase sequence. Amino acidpositions amenable to mutation in accordance with the present inventionare indicated with an asterisk (*).

FIG. 2 provides sequence identities among the following DNA Polymerase Ienzymes: Thermus sp. Z05 DNA polymerase (Z05); Thermus aquaticus DNApolymerase (Taq); Thermus filiformis DNA polymerase (Tfi); Thermusflavus DNA polymerase (Tfl); Thermus sp. Sps17 DNA polymerase (Sps17);Thermus thermophilus DNA polymerase (Tth); Thermus caldophilus DNApolymerase (Tca); Deinococcus radiodurans DNA polymerase (Dra);Thermotoga maritima DNA polymerase (Tma); Thermotoga neopolitana DNApolymerase (Tne); Thermosipho africanus DNA polymerase (Taf); Bacillusstearothermophilus DNA polymerase (Bst); and Bacillus caldotenax DNApolymerase (Bca). (A) sequence identities over the entire polymerase Ienzyme (corresponding to amino acids 1-834 of Z05); and (B) sequenceidentities over the polymerase sub domain corresponding to amino acids420-834 of Z05.

DETAILED DESCRIPTION

The present invention provides improved DNA polymerases in which one ormore amino acids in the polymerase domain have been identified asimproving one or more polymerase activity or characteristics. The DNApolymerases of the invention are active enzymes having increased3′-mismatch discrimination activity (i.e., the inventive polymerasesdescribed herein are less likely to extend primers that are mismatchedto template at or near the 3′ end of the primer) relative to theunmodified form of the polymerase otherwise identical except for theamino acid difference noted herein. The DNA polymerases are useful in avariety of applications involving polynucleotide extension oramplification of polynucleotide templates, including, for example,applications in recombinant DNA studies and medical diagnosis ofdisease.

Polymerases of the Invention

In some embodiments, the DNA polymerases of the invention can becharacterized by having the following motif:

-   -   Lys-Leu-X₁-X₂-Thr-X₃-X₄-X₅-X₆-X₇-X₈ (also referred to herein in        the one-letter code as K-L-X₁-X₂-T-X₃-X₄-X₅-X₆-X₇-X₈); wherein        -   X₁ is Lys (K), Arg (R) or Gln (Q);        -   X₂ is Ser (S), Asn (N) or Gly (G);        -   X₃ is any amino acid other than Tyr (Y);        -   X₄ is Val (V), Ile (I) or Leu (L);        -   X₅ is Asp (D) or Glu (E);        -   X₆ is Pro (P), Ala (A), Thr (T), Ser (S) or Gly (G);        -   X₇ is Leu (L) or Ile (I); and        -   X₈ is Pro (P) or Leu (L) (SEQ ID NO:8).

In some embodiments, X₃ is selected from G, A, L, M, W, P, S, T, F, C,N, Q, D, E, K, R, V, I or H (SEQ ID NO:42).

In some embodiments, DNA polymerases of the invention can becharacterized by having the following motif (corresponding to Thermusand Thermotoga):

-   -   Lys-Leu-Lys-X₂-Thr-X₃-X₄-Asp-X₆-Leu-Pro (also referred to herein        in the one-letter code as K-L-K-X₂-T-X₃-X₄-D-X₆-L-P); wherein    -   X₂ is Asn (N) or Ser (S);    -   X₃ is any amino acid other than Tyr (Y);    -   X₄ is Val (V) or Ile (I); and    -   X₆ is Pro (P) or Ala (A) (SEQ ID NO:9).

In some embodiments, DNA polymerases of the invention can becharacterized by having the following motif:

-   -   Lys-Leu-Lys-X₂-Thr-X₃-X₄-Asp-Pro-Leu-Pro (also referred to        herein in the one-letter code as K-L-K-X₂-T-X₃-X₄-D-P-L-P);        wherein    -   X₂ is Asn (N) or Ser (5);    -   X₃ is any amino acid other than Tyr (Y); and    -   X₄ is Val (V) or Ile (I) (SEQ ID NO:10).

In some embodiments, DNA polymerases of the invention can becharacterized by having the following motif:

-   -   Lys-Leu-Lys-X₂-Thr-X₃-X₄-Asp-Pro-Leu-Pro (also referred to        herein in the one-letter code as K-L-K-X₂-T-X₃-X₄-D-P-L-P);        wherein    -   X₂ is Asn (N) or Ser (S);    -   X₃ is Cys (C) or Asn (N); and    -   X₄ is Val (V) or Ile (I) (SEQ ID NO:11).

This motif is present within the “fingers” domain of many Family A typeDNA-dependent DNA polymerases, particularly thermostable DNA polymerasesfrom thermophilic bacteria (Li et al., EMBO J. 17:7514-7525, 1998). Forexample, FIG. 1 shows an amino acid sequence alignment comprising thenative sequence corresponding to the motif above in DNA polymerases fromseveral species of bacteria: Escherichia coli, Bacillus caldotenax,Bacillus stearothermophilus, Deinococcus radiodurans, Thermosiphoafricanus, Thermotoga maritima, Thermotoga neopolitana, Thermusaquaticus, Thermus caldophilus, Thermus filiformus, Thermus flavus,Thermus sp. Sps17, Thermus sp. Z05, and Thermus thermophilus. As shown,the motif of SEQ ID NO:8 (except where X₃ is Y) is present in each ofthese polymerases, indicating a conserved function for this region ofthe polymerase. FIG. 2 provides sequence identities among these DNApolymerases.

Accordingly, in some embodiments, the invention provides for apolymerase comprising SEQ ID NO:8, 9, 10, or 11 (e.g., where X₃ isselected from G, A, L, M, W, P, S, T, F, C, N, Q, D, E, K, R, V, I orH), having the improved activity and/or characteristics describedherein, and wherein the DNA polymerase is otherwise a wild-type or anaturally occurring DNA polymerase, such as, for example, a polymerasefrom any of the species of thermophilic bacteria listed above, or issubstantially identical to such a wild-type or a naturally occurring DNApolymerase. For example, in some embodiments, the polymerase of theinvention comprises SEQ ID NO:8, 9, 10, or 11 and is at least 80%, 85%,90%, or 95% identical to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 36, 37, 38, 39,40, or 41. In one variation, the unmodified form of the polymerase isfrom a species of the genus Thermus. In some embodiments of theinvention, the unmodified polymerase is from a thermophilic speciesother than Thermus, e.g., Thermotoga. The full nucleic acid and aminoacid sequence for numerous thermostable DNA polymerases are available.The sequences each of Thermus aquaticus (Taq) (SEQ ID NO:2), Thermusthermophilus (Tth) (SEQ ID NO:6), Thermus species Z05 (SEQ ID NO:1),Thermus species Sps17 (SEQ ID NO:5), Thermotoga maritima (Tma) (SEQ IDNO:38), and Thermosipho africanus (Taf) (SEQ ID NO:37) polymerase havebeen published in PCT International Patent Publication No. WO 92/06200,which is incorporated herein by reference. The sequence for the DNApolymerase from Thermus flavus (SEQ ID NO:4) has been published inAkhmetzjanov and Vakhitov (Nucleic Acids Research 20:5839, 1992), whichis incorporated herein by reference. The sequence of the thermostableDNA polymerase from Thermus caldophilus (SEQ ID NO:7) is found inEMBL/GenBank Accession No. U62584. The sequence of the thermostable DNApolymerase from Thermus filiformis can be recovered from ATCC DepositNo. 42380 using, e.g., the methods provided in U.S. Pat. No. 4,889,818,as well as the sequence information provided in Table 1. The sequence ofthe Thermotoga neapolitana DNA polymerase (SEQ ID NO:39) is from GeneSeqPatent Data Base Accession No. R98144 and PCT WO 97/09451, eachincorporated herein by reference. The sequence of the thermostable DNApolymerase from Bacillus caldotenax (SEQ ID NO:41) is described in,e.g., Uemori et al. (J Biochem (Tokyo) 113(3):401-410, 1993; see also,Swiss-Prot database Accession No. Q04957 and GenBank Accession Nos.D12982 and BAA02361), which are each incorporated by reference. Examplesof unmodified forms of DNA polymerases that can be modified as describedherein are also described in, e.g., U.S. Pat. Nos. 6,228,628, entitled“Mutant chimeric DNA polymerase” issued May 8, 2001 to Gelfand et al.;6,346,379, entitled “Thermostable DNA polymerases incorporatingnucleoside triphosphates labeled with fluorescein family dyes” issuedFeb. 12, 2002 to Gelfand et al.; 7,030,220, entitled “Thermostableenzyme promoting the fidelity of thermostable DNA polymerases—forimprovement of nucleic acid synthesis and amplification in vitro” issuedApr. 18, 2006 to Ankenbauer et al.; 6,881,559, entitled “Mutant B-typeDNA polymerases exhibiting improved performance in PCR” issued Apr. 19,2005 to Sobek et al.; 6,794,177, entitled “Modified DNA-polymerase fromcarboxydothermus hydrogenofonnans and its use for coupled reversetranscription and polymerase chain reaction” issued Sep. 21, 2004 toMarkau et al.; 6,468,775, entitled “Thermostable DNA polymerase fromcarboxydothermus hydrogenoformans” issued Oct. 22, 2002 to Ankenbauer etal.; and U.S. Pat. Appl. Nos. 20040005599, entitled “Thermostable orthermoactive DNA polymerase molecules with attenuated 3′-5′ exonucleaseactivity” filed Mar. 26, 2003 by Schoenbrunner et al.; 20020012970,entitled “High temperature reverse transcription using mutant DNApolymerases” filed Mar. 30, 2001 by Smith et al.; 20060078928, entitled“Thermostable enzyme promoting the fidelity of thermostable DNApolymerases—for improvement of nucleic acid synthesis and amplificationin vitro” filed Sep. 29, 2005 by Ankenbauer et al.; 20040115639,entitled “Reversibly modified thermostable enzymes for DNA synthesis andamplification in vitro” filed Dec. 11, 2002 by Sobek et al., which areeach incorporated by reference. Representative full length polymerasesequences are also provided in the sequence listing.

In some embodiments, the polymerase of the invention, as well as havinga polymerase domain comprising SEQ ID NOS:8, 9, 10, or 11, alsocomprises a nuclease domain (e.g., corresponding to positions 1 to 291of Z05).

In some embodiments, a polymerase of the invention is a chimericpolymerase, i.e., comprising polypeptide regions from two or moreenzymes. Examples of such chimeric DNA polymerases are described in,e.g., U.S. Pat. No. 6,228,628, which is incorporated by reference hereinin its entirety. Particularly suitable are chimeric CS-family DNApolymerases, which include the CS5 (SEQ ID NO:29) and CS6 (SEQ ID NO:30)polymerases and variants thereof having substantial sequence identity orsimilarity to SEQ ID NO:29 or SEQ ID NO:30 (typically at least 80%sequence identity and more typically at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity) and can thus be modified tocontain SEQ ID NO:8.

The CS5 and CS6 DNA polymerases are chimeric enzymes derived fromThermus sp. Z05 and Thermotoga maritima (Tma) DNA polymerases. Theycomprise the N-terminal 5′-nuclease domain of the Thermus enzyme and theC-terminal 3′-5′ exonuclease and the polymerase domains of the Tmaenzyme. These enzymes have efficient reverse transcriptase activity, canextend nucleotide analog-containing primers, and can utilizealpha-phosphorothioate dNTPs, dUTP, dITP, and also fluorescein- andcyanine-dye family labeled dNTPs. The CS5 and CS6 polymerases are alsoefficient Mg²⁺-activated PCR enzymes. The CS5 and CS6 chimericpolymerases are further described in, e.g., U.S. Pat. ApplicationPublication No. 2004/0005599, which is incorporated by reference hereinin its entirety.

In some embodiments, the polymerase of the invention comprises SEQ IDNO:8, 9, 10, or 11 and further comprises one or more additional aminoacid changes (e.g., by amino acid substitution, addition, or deletion)compared to a native polymerase. In some embodiments, such polymerasesretain the amino acid motif of SEQ ID NO:8 (or a motif of SEQ ID NO:9,10 or 11), and further comprise the amino acid motif of SEQ ID NO:27(corresponding to the D580X mutation of Z05 (SEQ ID NO:1)) as follows:

-   -   T-G-R-L-S—S—X₇-X₈-P-N-L-Q-N; wherein    -   X₇ is Ser (S) or Thr (T); and    -   X₈ is any amino acid other than D or E (SEQ ID NO:27)        The mutation characterized by SEQ ID NO:27 is discussed in more        detail in, e.g., US Patent Publication No. 2009/0148891. In some        embodiments, such functional variant polymerases typically will        have substantial sequence identity or similarity to the        wild-type or naturally occurring polymerase (e.g., SEQ ID NO:1,        2, 3, 4, 5, 6, 7, 39, 40, 41, 42, 43, or 44), typically at least        80% sequence identity and more typically at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In some embodiments, the amino acid at position X₃ is substituted withan amino acid as set forth in SEQ ID NO:8, 9, 10 or 11, and the aminoacid at position X₈ is substituted with an amino acid as set forth inSEQ ID NO:27. Thus, in some embodiments, the amino acid at position X₃is any amino acid other than Tyr (Y) and the amino acid at position X₈is any amino acid other than Asp (D) or Glu (E). In some embodiments,amino acid substitutions include Leucine (L), Glycine (G), Threonine(T), Glutamine (Q), Alanine (A), Serine (S), Asparagine (N), Arginine(R), and Lysine (K) at position X₈ of SEQ ID NO:27. In certainembodiments, amino acid substitutions independently include Cysteine (C)or Asparagine (N) at position X₃, and Glycine (G) at position X₈. Othersuitable amino acid substitution(s) at one or more of the identifiedsites can be determined using, e.g., known methods of site-directedmutagenesis and determination of polynucleotide extension performance inassays described further herein or otherwise known to persons of skillin the art.

Because the precise length of DNA polymerases vary, the precise aminoacid positions corresponding to each of X₃ and X₈ can vary depending onthe particular polymerase used. Amino acid and nucleic acid sequencealignment programs are readily available (see, e.g., those referred tosupra) and, given the particular motifs identified herein, serve toassist in the identification of the exact amino acids (and correspondingcodons) for modification in accordance with the present invention. Thepositions corresponding to each of X₃ and X₈ are shown in Table 1 forrepresentative chimeric thermostable DNA polymerases and thermostableDNA polymerases from exemplary thermophilic species.

TABLE 1 Amino Acid Positions Corresponding to Motif Positions X₃ (e.g.,of SEQ ID NOS: 8, 9, 10, and 11) and X₈ (of SEQ ID NO: 27) in ExemplaryPolymerases. Orsanism or Chimeric Sequence Amino Acid Position Consensus(SEQ ID NO:) X₃ X₈ (of SEQ ID NO: 27) T. thermophilus (6) 547 580 T.caldophilus (7) 547 580 T. sp. Z05 (1) 547 580 T. aquaticus (2) 545 578T. flavus (4) 544 577 T. filiformis (3) 543 576 T. sp. Sps17 (5) 543 576T. maritima (38) 607 640 T. neapolitana (39) 607 640 T. africanus (37)606 639 B. caldotenax (41) 588 621 B. stearothermophilus (40) 587 620CS5 (29) 607 640 CS6 (30) 607 640

In some embodiments, the mutant DNA polymerase of the present inventionis derived from Thermus sp. Z05 DNA polymerase (SEQ ID NO:1) or avariant thereof (e.g., D580G or the like). As referred to above, inThermus sp. Z05 DNA polymerase, position X₃ corresponds to Tyrosine (Y)at position 547, and position X₈ corresponds to Aspartate (D) atposition 580. Thus, in certain variations of the invention, the mutantpolymerase comprises at least one amino acid substitution, relative to aThermus sp. Z05 DNA polymerase, at Y547 and D580. Thus, typically, theamino acid at position Y547, is not Y. In some embodiments, the aminoacid at position 547 is selected from G, A, V, L, I, M, F, W, P, S, T,C, N, Q, D, E, K, R or H. In certain embodiments, the amino acid residueat position Y547 is C or N. Amino acid residues at position D580 can beselected from Leucine (L), Glycine (G), Threonine (T), Glutamine (Q),Alanine (A), Serine (S), Asparagine (N), Arginine (R), and Lysine (K).In certain embodiments, the amino acid residue at position D580 is G.

In some embodiments, the Z05 DNA polymerase further comprises additionalamino acid substitutions. For example, in some embodiments, the aminoacid at position 475 of SEQ ID NO:1 is any amino acid other than E. Insome embodiments, the amino acid at position 475 of SEQ ID NO:1 isselected from G, A, V, L, I, M, F, W, P, S, T, C, N, Q, D, Y, K, R or H.In some embodiments, the amino acid at position 475 of SEQ ID NO:1 is D.In some embodiments, the amino acid at position 632 of SEQ ID NO:1 isany amino acid other than R. In some embodiments, the amino acid atposition 632 of SEQ ID NO:1 is selected from G, A, V, L, I, M, F, W, P,S, T, C, N, Q, D, Y, K, E or H. In some embodiments, the amino acid atposition 632 of SEQ ID NO:1 is S.

Exemplary Thermus sp. Z05 DNA polymerase mutants include thosecomprising the amino acid substitution(s) Y547C or Y547N in combinationwith E475D, R632S, and/or D580G. In some embodiments, the mutant Thermussp. Z05 DNA polymerase comprises, e.g., amino acid residue substitutionsY547C and D580G; or Y547N and D580G. In some embodiments, the mutantThermus sp. Z05 DNA polymerase comprises, e.g., amino acid residuesubstitutions (a) Y547C, E475D, and D580G; or (b) Y547N, E475D, andD580G. In some embodiments, the mutant Thermus sp. Z05 DNA polymerasecomprises, e.g., amino acid residue substitutions (a) Y547C, R632S, andD580G; or (b) Y547N, R632S, and D580G. In some embodiments, the mutantThermus sp. Z05 DNA polymerase comprises, e.g., amino acid residuesubstitutions (a) Y547C, E475D, and R632S; or (b) Y547N, E475D, andR632S. In some embodiments, the mutant Thermus sp. Z05 DNA polymerasecomprises, e.g., amino acid residue substitutions (a) Y547C, E475D,R632S, and D580G; or (b) Y547N, E475D, R632S, and D580G.

In some embodiments, the amino acid at the position corresponding toposition 547 of SEQ ID NO:1 is an amino acid having a non-polar,uncharged side-chain (e.g., G, A, L, M, W, P, F, C, V, or I) or a polar,uncharged side-chain (other than Y, e.g., N, Q, H, S, or T) at neutralpH (e.g., about pH 7.4). In some embodiments, the DNA polymerase of theinvention is derived from a Thermus species, and the amino acid at theposition corresponding to position 547 of SEQ ID NO:1 is an amino acidhaving a non-polar, uncharged side-chain (e.g., G, A, L, M, W, P, F, C,V, or I) at neutral pH. In some embodiments, the DNA polymerase of theinvention is derived from a Thermus species, and the amino acid at theposition corresponding to position 547 of SEQ ID NO:1 is an amino acidhaving a polar, uncharged side-chain (other than Y, e.g., N, Q, H, S, orT) at neutral pH. In some embodiments, the amino acid at the positioncorresponding to position 547 of SEQ ID NO:1 having a non-polar,uncharged side-chain is C. In some embodiments, the amino acid at theposition corresponding to position 547 of SEQ ID NO:1 having a polar,uncharged side-chain is S.

In some embodiments, the DNA polymerases of the present invention canalso include other, non-substitutional modification(s). Suchmodifications can include, for example, covalent modifications known inthe art to confer an additional advantage in applications comprisingpolynucleotide extension. For example, in certain embodiments, themutant DNA polymerase further includes a thermally reversible covalentmodification. DNA polymerases comprising such thermally reversiblemodifications are particularly suitable for hot-start applications, suchas, e.g., various hot-start PCR techniques. Thermally reversiblemodifier reagents amenable to use in accordance with the mutant DNApolymerases of the present invention are described in, for example, U.S.Pat. No. 5,773,258 to Birch et al., which is incorporated by referenceherein.

For example, particularly suitable polymerases comprising a thermallyreversible covalent modification are produced by a reaction, carried outat alkaline pH at a temperature which is less than about 25° C.; of amixture of a thermostable enzyme and a dicarboxylic acid anhydridehaving a general formula as set forth in the following formula I:

where R₁ and R₂ are hydrogen or organic radicals, which may be linked;or having the following formula II:

where R₁ and R₂ are organic radicals, which may linked, and thehydrogens are cis, essentially as described in Birch et al, supra.

The DNA polymerases of the present invention can be constructed bymutating the DNA sequences that encode the corresponding unmodifiedpolymerase (e.g., a wild-type polymerase or a corresponding variant fromwhich the polymerase of the invention is derived), such as by usingtechniques commonly referred to as site-directed mutagenesis. Nucleicacid molecules encoding the unmodified form of the polymerase can bemutated by a variety of polymerase chain reaction (PCR) techniqueswell-known to one of ordinary skill in the art. (See, e.g., PCRStrategies (M. A. Innis, D. H. Gelfand, and J. J. Sninsky eds., 1995,Academic Press, San Diego, Calif.) at Chapter 14; PCR Protocols: A Guideto Methods and Applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky,and T. J. White eds., Academic Press, NY, 1990).

By way of non-limiting example, the two primer system, utilized in theTransformer Site-Directed Mutagenesis kit from Clontech, may be employedfor introducing site-directed mutants into a polynucleotide encoding anunmodified form of the polymerase. Following denaturation of the targetplasmid in this system, two primers are simultaneously annealed to theplasmid; one of these primers contains the desired site-directedmutation, the other contains a mutation at another point in the plasmidresulting in elimination of a restriction site. Second strand synthesisis then carried out, tightly linking these two mutations, and theresulting plasmids are transformed into a mutS strain of E. coli.Plasmid DNA is isolated from the transformed bacteria, restricted withthe relevant restriction enzyme (thereby linearizing the unmutatedplasmids), and then retransformed into E. coli. This system allows forgeneration of mutations directly in an expression plasmid, without thenecessity of subcloning or generation of single-stranded phagemids. Thetight linkage of the two mutations and the subsequent linearization ofunmutated plasmids result in high mutation efficiency and allow minimalscreening. Following synthesis of the initial restriction site primer,this method requires the use of only one new primer type per mutationsite. Rather than prepare each positional mutant separately, a set of“designed degenerate” oligonucleotide primers can be synthesized inorder to introduce all of the desired mutations at a given sitesimultaneously. Transformants can be screened by sequencing the plasmidDNA through the mutagenized region to identify and sort mutant clones.Each mutant DNA can then be restricted and analyzed by electrophoresis,such as for example, on a Mutation Detection Enhancement gel(Mallinckrodt Baker, Inc., Phillipsburg, N.J.) to confirm that no otheralterations in the sequence have occurred (by band shift comparison tothe unmutagenized control). Alternatively, the entire DNA region can besequenced to confirm that no additional mutational events have occurredoutside of the targeted region.

Verified mutant duplexes in pET (or other) overexpression vectors can beemployed to transform E. coli such as, e.g., strain E. coli BL21 (DE3)pLysS, for high level production of the mutant protein, and purificationby standard protocols. The method of FAB-MS mapping, for example, can beemployed to rapidly check the fidelity of mutant expression. Thistechnique provides for sequencing segments throughout the whole proteinand provides the necessary confidence in the sequence assignment. In amapping experiment of this type, protein is digested with a protease(the choice will depend on the specific region to be modified since thissegment is of prime interest and the remaining map should be identicalto the map of unmutagenized protein). The set of cleavage fragments isfractionated by, for example, microbore HPLC (reversed phase or ionexchange, again depending on the specific region to be modified) toprovide several peptides in each fraction, and the molecular weights ofthe peptides are determined by standard methods, such as FAB-MS. Thedetermined mass of each fragment are then compared to the molecularweights of peptides expected from the digestion of the predictedsequence, and the correctness of the sequence quickly ascertained. Sincethis mutagenesis approach to protein modification is directed,sequencing of the altered peptide should not be necessary if the MS dataagrees with prediction. If necessary to verify a changed residue,CAD-tandem MS/MS can be employed to sequence the peptides of the mixturein question, or the target peptide can be purified for subtractive Edmandegradation or carboxypeptidase Y digestion depending on the location ofthe modification.

Mutant DNA polymerases with more than one amino acid substituted can begenerated in various ways. In the case of amino acids located closetogether in the polypeptide chain, they may be mutated simultaneouslyusing one oligonucleotide that codes for all of the desired amino acidsubstitutions. If however, the amino acids are located some distancefrom each other (separated by more than ten amino acids, for example) itis more difficult to generate a single oligonucleotide that encodes allof the desired changes. Instead, one of two alternative methods may beemployed. In the first method, a separate oligonucleotide is generatedfor each amino acid to be substituted. The oligonucleotides are thenannealed to the single-stranded template DNA simultaneously, and thesecond strand of DNA that is synthesized from the template will encodeall of the desired amino acid substitutions. An alternative methodinvolves two or more rounds of mutagenesis to produce the desiredmutant. The first round is as described for the single mutants: DNAencoding the unmodified polymerase is used for the template, anoligonucleotide encoding the first desired amino acid substitution(s) isannealed to this template, and the heteroduplex DNA molecule is thengenerated. The second round of mutagenesis utilizes the mutated DNAproduced in the first round of mutagenesis as the template. Thus, thistemplate already contains one or more mutations. The oligonucleotideencoding the additional desired amino acid substitution(s) is thenannealed to this template, and the resulting strand of DNA now encodesmutations from both the first and second rounds of mutagenesis. Thisresultant DNA can be used as a template in a third round of mutagenesis,and so on. Alternatively, the multi-site mutagenesis method of Seyfang &Jin (Anal. Biochem. 324:285-291. 2004) may be utilized.

Accordingly, also provided are recombinant nucleic acids encoding any ofthe DNA polymerases of the present invention (e.g., polymerasescomprising any of SEQ ID NOS:8, 9, 10, or 11). Using a nucleic acid ofthe present invention, encoding a DNA polymerase of the invention, avariety of vectors can be made. Any vector containing replicon andcontrol sequences that are derived from a species compatible with thehost cell can be used in the practice of the invention. Generally,expression vectors include transcriptional and translational regulatorynucleic acid regions operably linked to the nucleic acid encoding themutant DNA polymerase. The term “control sequences” refers to DNAsequences necessary for the expression of an operably linked codingsequence in a particular host organism. The control sequences that aresuitable for prokaryotes, for example, include a promoter, optionally anoperator sequence, and a ribosome binding site. In addition, the vectormay contain a Positive Retroregulatory Element (PRE) to enhance thehalf-life of the transcribed mRNA (see Gelfand et al. U.S. Pat. No.4,666,848). The transcriptional and translational regulatory nucleicacid regions will generally be appropriate to the host cell used toexpress the polymerase. Numerous types of appropriate expressionvectors, and suitable regulatory sequences are known in the art for avariety of host cells. In general, the transcriptional and translationalregulatory sequences may include, e.g., promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. Intypical embodiments, the regulatory sequences include a promoter andtranscriptional start and stop sequences. Vectors also typically includea polylinker region containing several restriction sites for insertionof foreign DNA. In certain embodiments, “fusion flags” are used tofacilitate purification and, if desired, subsequent removal of tag/flagsequence, e.g., “His-Tag”. However, these are generally unnecessary whenpurifying an thermoactive and/or thermostable protein from a mesophilichost (e.g., E. coli) where a “heat-step” may be employed. Theconstruction of suitable vectors containing DNA encoding replicationsequences, regulatory sequences, phenotypic selection genes, and themutant polymerase of interest are prepared using standard recombinantDNA procedures. Isolated plasmids, viral vectors, and DNA fragments arecleaved, tailored, and ligated together in a specific order to generatethe desired vectors, as is well-known in the art (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory Press, New York, N.Y., 2nd ed. 1989)).

In certain embodiments, the expression vector contains a selectablemarker gene to allow the selection of transformed host cells. Selectiongenes are well known in the art and will vary with the host cell used.Suitable selection genes can include, for example, genes coding forampicillin and/or tetracycline resistance, which enables cellstransformed with these vectors to grow in the presence of theseantibiotics.

In one aspect of the present invention, a nucleic acid encoding a DNApolymerase of the invention is introduced into a cell, either alone orin combination with a vector. By “introduced into” or grammaticalequivalents herein is meant that the nucleic acids enter the cells in amanner suitable for subsequent integration, amplification, and/orexpression of the nucleic acid. The method of introduction is largelydictated by the targeted cell type. Exemplary methods include CaPO₄precipitation, liposome fusion, LIPOFECTIN®, electroporation, viralinfection, and the like.

In some embodiments, prokaryotes are used as host cells for the initialcloning steps of the present invention. They are particularly useful forrapid production of large amounts of DNA, for production ofsingle-stranded DNA templates used for site-directed mutagenesis, forscreening many mutants simultaneously, and for DNA sequencing of themutants generated. Suitable prokaryotic host cells include E. coli K12strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325), E.coli K12 strain DG116 (ATCC No. 53,606), E. coli X1776 (ATCC No.31,537), and E. coli B; however many other strains of E. coli, such asHB101, JM101, NM522, NM538, NM539, and many other species and genera ofprokaryotes including bacilli such as Bacillus subtilis, otherenterobacteriaceae such as Salmonella typhimurium or Serratia marcesans,and various Pseudomonas species can all be used as hosts. Prokaryotichost cells or other host cells with rigid cell walls are typicallytransformed using the calcium chloride method as described in section1.82 of Sambrook et al., supra. Alternatively, electroporation can beused for transformation of these cells. Prokaryote transformationtechniques are set forth in, for example Dower, in Genetic Engineering,Principles and Methods 12:275-296 (Plenum Publishing Corp., 1990);Hanahan et al., Meth. Enzymol., 204:63, 1991. Plasmids typically usedfor transformation of E. coli include pBR322, pUCI8, pUCI9, pUCI18,pUC119, and Bluescript M13, all of which are described in sections1.12-1.20 of Sambrook et al., supra. However, many other suitablevectors are available as well.

In some embodiments, the DNA polymerases of the present invention areproduced by culturing a host cell transformed with an expression vectorcontaining a nucleic acid encoding the DNA polymerase, under theappropriate conditions to induce or cause expression of the DNApolymerase. Methods of culturing transformed host cells under conditionssuitable for protein expression are well-known in the art (see, e.g.,Sambrook et al., supra). Suitable host cells for production of thepolymerases from lambda pL promotor-containing plasmid vectors includeE. coli strain DG116 (ATCC No. 53606) (see U.S. Pat. No. 5,079,352 andLawyer, F. C. et al., PCR Methods and Applications 2:275-87, 1993, whichare both incorporated herein by reference). Following expression, thepolymerase can be harvested and isolated. Methods for purifying thethermostable DNA polymerase are described in, for example, Lawyer etal., supra.

Once purified, a DNA polymerase's 3′ mismatch discrimination can beassayed. For example, in some embodiments, 3′ mismatch discriminationactivity is determined by comparing the amplification of a targetsequence perfectly matched to the primer to amplification of a targetthat has a single base mismatch at the 3′ end of the primer.Amplification can be detected, for example, in real time by use ofTaqMan™ probes. Ability of a polymerase to distinguish between the twotarget sequences can be estimated by comparing the Cps of the tworeactions. Optionally, simultaneous amplification of a second targetgene in each well can be performed and detected in a second opticalchannel as a control. “Delta Cp values” refer to the difference in valuebetween the Cp associated with the mismatched template minus the Cp ofthe matched target (see, e.g., the Examples). In some embodiments, theimproved polymerases of the invention have a delta Cp value of at least1, 2, 3, 4, 5, or more compared to an otherwise identical controlpolymerase having a native amino acid (e.g., Y) at position X₃ of SEQ IDNO:8. In some embodiments, this determination is made with the precisematerials and conditions set forth in the Examples.

Methods of the Invention

The improved DNA polymerases of the present invention may be used forany purpose in which such enzyme activity is necessary or desired. Theimproved DNA polymerase can be a thermoactive or thermostable DNApolymerase, as described herein. Accordingly, in one aspect of theinvention, methods of polynucleotide extension, including PCR, using thepolymerases of the invention are provided. In some embodiments, theinvention provides a thermoactive DNA polymerase that is useful toextend an RNA or DNA template when amplification of the template nucleicacid is not required, for example, when it is desired to immediatelydetect the presence of a target nucleic acid. In some embodiments, theinvention provides a thermostable DNA polymerase that is useful when itis desired to extend and/or amplify a target nucleic acid. Conditionssuitable for polynucleotide extension are known in the art. (See, e.g.,Sambrook et al., supra. See also Ausubel et al., Short Protocols inMolecular Biology (4th ed., John Wiley & Sons 1999). Generally, a primeris annealed, i.e., hybridized, to a target nucleic acid to form aprimer-template complex. The primer-template complex is contacted withthe mutant DNA polymerase and nucleoside triphosphates in a suitableenvironment to permit the addition of one or more nucleotides to the 3′end of the primer, thereby producing an extended primer complementary tothe target nucleic acid. The primer can include, e.g., one or morenucleotide analog(s). In addition, the nucleoside triphosphates can beconventional nucleotides, unconventional nucleotides (e.g.,ribonucleotides or labeled nucleotides), or a mixture thereof. In somevariations, the polynucleotide extension reaction comprisesamplification of a target nucleic acid. Conditions suitable for nucleicacid amplification using a DNA polymerase and a primer pair are alsoknown in the art (e.g., PCR amplification methods). (See, e.g., Sambrooket al., supra; Ausubel et al., supra; PCR Applications: Protocols forFunctional Genomics (Innis et al. eds., Academic Press 1999).

In some embodiments, use of the present polymerases, which provideincreased 3′ mismatch discrimination, allow for, e.g., rare alleledetection. For example, the fidelity of 3′ mismatch discrimination of aparticular polymerase sets its sensitivity (ability to accurately detectsmall quantities of a target sequence in the presence of largerquantities of a different but related non-target sequence). Thus,increased 3′-mismatch discrimination results in greater sensitivity fordetection of rare alleles. Rare allele detection is useful, for example,when screening biopsies or other samples for rare genetic changes, e.g.,a cell carrying a cancer allele in a mass of normal cells.

In some embodiments, the improved polymerases are used forpolynucleotide extension in the context of allele specific PCR or singlenucleotide polymorphism (SNP) detection. Exemplary SNP detection methodsare described in Chen et al., “Single nucleotide polymorphismgenotyping: biochemistry, protocol, cost and throughput”Pharmacogenomics J. 3(2):77-96 (2003); Kwok et al., “Detection of singlenucleotide polymorphisms” Curr. Issues Mol. Biol. 5(2):43-60 (April2003); Shi, “Technologies for individual genotyping: detection ofgenetic polymorphisms in drug targets and disease genes” Am. J.Pharmacogenomics 2(3):197-205 (2002); and Kwok, “Methods for genotypingsingle nucleotide polymorphisms” Annu. Rev. Genomics Hum. Genet.2:235-58 (2001). Exemplary techniques for high-throughput SNP detectionare described in Marnellos, “High-throughput SNP analysis for geneticassociation studies” Curr. Opin. Drug Discov. Devel. 6(3):317-21 (May2003). Common SNP detection methods include, but are not limited to,TaqMan assays, molecular beacon assays, nucleic acid arrays,allele-specific primer extension, allele-specific PCR, arrayed primerextension, homogeneous primer extension assays, primer extension withdetection by mass spectrometry, pyrosequencing, multiplex primerextension sorted on genetic arrays, ligation with rolling circleamplification, homogeneous ligation, OLA (U.S. Pat. No. 4,988,167),multiplex ligation reaction sorted on genetic arrays,restriction-fragment length polymorphism, single base extension-tagassays, and the Invader assay. Such methods may be used in combinationwith detection mechanisms such as, for example, luminescence orchemiluminescence detection, fluorescence detection, time-resolvedfluorescence detection, fluorescence resonance energy transfer,fluorescence polarization, mass spectrometry, and electrical detection.

Detection of multiple different alleles can also be accomplished usingmultiplex reactions, which allow the detection of multiple differentalleles in a single reaction. In multiplex reactions, two or moreallele-specific primers are used to extend and amplify SNPs or multiplenucleotide polymorphisms or alleles. Exemplary methods for multiplexdetection of single and multiple nucleotide polymorphisms are describedin U.S. Patent Publication No. 2006/0172324, the contents of which areexpressly incorporated by reference herein in its entirety.

Other methods for detecting extension products or amplification productsusing the improved polymerases described herein include the use offluorescent double-stranded nucleotide binding dyes or fluorescentdouble-stranded nucleotide intercalating dyes. Examples of fluorescentdouble-stranded DNA binding dyes include SYBR-green (Molecular Probes).Examples of fluorescent double-stranded intercalating dyes includeethidium bromide. The double stranded DNA binding dyes can be used inconjunction with melting curve analysis to measure primer extensionproducts and/or amplification products. The melting curve analysis canbe performed on a real-time PCR instrument, such as the ABI 5700/7000(96 well format) or ABI 7900 (384 well format) instrument with onboardsoftware (SDS 2.1). Alternatively, the melting curve analysis can beperformed as an end point analysis. Exemplary methods of melting pointanalysis are described in U.S. Patent Publication No. 2006/0172324, thecontents of which are expressly incorporated by reference herein in itsentirety.

In yet other embodiments, the polymerases of the invention are used forprimer extension in the context of DNA sequencing, DNA labeling, orlabeling of primer extension products. For example, DNA sequencing bythe Sanger dideoxynucleotide method (Sanger et al., Proc. Natl. Acad.Sci. USA 74: 5463, 1977) is improved by the present invention forpolymerases capable of incorporating unconventional, chain-terminatingnucleotides. Advances in the basic Sanger et al. method have providednovel vectors (Yanisch-Perron et al., Gene 33:103-119, 1985) and baseanalogues (Mills et al., Proc. Natl. Acad. Sci. USA 76:2232-2235, 1979;and Barr et al., Biotechniques 4:428-432, 1986). In general, DNAsequencing requires template-dependent primer extension in the presenceof chain-terminating base analogs, resulting in a distribution ofpartial fragments that are subsequently separated by size. The basicdideoxy sequencing procedure involves (i) annealing an oligonucleotideprimer, optionally labeled, to a template; (ii) extending the primerwith DNA polymerase in four separate reactions, each containing amixture of unlabeled dNTPs and a limiting amount of one chainterminating agent such as a ddNTP, optionally labeled; and (iii)resolving the four sets of reaction products on a high-resolutiondenaturing polyacrylamide/urea gel. The reaction products can bedetected in the gel by autoradiography or by fluorescence detection,depending on the label used, and the image can be examined to infer thenucleotide sequence. These methods utilize DNA polymerase such as theKlenow fragment of E. coli Pol I or a modified T7 DNA polymerase.

The availability of thermostable polymerases, such as Taq DNApolymerase, resulted in improved methods for sequencing withthermostable DNA polymerase (see Innis et al., Proc. Natl. Acad. Sci.USA 85:9436, 1988) and modifications thereof referred to as “cyclesequencing” (Murray, Nuc Acids Res. 17:8889, 1989). Accordingly,thermostable polymerases of the present invention can be used inconjunction with such methods. As an alternative to basic dideoxysequencing, cycle sequencing is a linear, asymmetric amplification oftarget sequences complementary to the template sequence in the presenceof chain terminators. A single cycle produces a family of extensionproducts of all possible lengths. Following denaturation of theextension reaction product from the DNA template, multiple cycles ofprimer annealing and primer extension occur in the presence ofterminators such as ddNTPs. Cycle sequencing requires less template DNAthan conventional chain-termination sequencing. Thermostable DNApolymerases have several advantages in cycle sequencing; they toleratethe stringent annealing temperatures which are required for specifichybridization of primer to nucleic acid targets as well as toleratingthe multiple cycles of high temperature denaturation which occur in eachcycle, e.g., 90-95° C. For this reason, AMPLITAQ® DNA Polymerase and itsderivatives and descendants, e.g., AmpliTaq CS DNA Polymerase andAmpliTaq FS DNA Polymerase have been included in Taq cycle sequencingkits commercialized by companies such as Perkin-Elmer (Norwalk, Conn.)and Applied Biosystems (Foster City, Calif.).

The improved polymerases find use in 454 sequencing (Roche) (Margulies,M et al. 2005, Nature, 437, 376-380). 454 sequencing involves two steps.In the first step, DNA is sheared into fragments of approximately300-800 base pairs, and the fragments are blunt ended. Oligonucleotideadaptors are then ligated to the ends of the fragments. The adaptorsserve as primers for amplification and sequencing of the fragments. Thefragments can be attached to DNA capture beads, e.g.,streptavidin-coated beads using, e.g., Adaptor B, which contains5′-biotin tag. The fragments attached to the beads are PCR amplifiedwithin droplets of an oil-water emulsion. The result is multiple copiesof clonally amplified DNA fragments on each bead. In the second step,the beads are captured in wells (pico-liter sized). Pyrosequencing isperformed on each DNA fragment in parallel. Addition of one or morenucleotides generates a light signal that is recorded by a CCD camera ina sequencing instrument. The signal strength is proportional to thenumber of nucleotides incorporated.

Pyrosequencing makes use of pyrophosphate (PPi) which is released uponnucleotide addition. PPi is converted to ATP by ATP sulfurylase in thepresence of adenosine 5′ phosphosulfate. Luciferase uses ATP to convertluciferin to oxyluciferin, and this reaction generates light that isdetected and analyzed.

Variations of chain termination sequencing methods include dye-primersequencing and dye-terminator sequencing. In dye-primer sequencing, theddNTP terminators are unlabeled, and a labeled primer is utilized todetect extension products (Smith et al., Nature 32:674-679, 1986). Indye-terminator DNA sequencing, a DNA polymerase is used to incorporatedNTPs and fluorescently labeled ddNTPs onto the end of a DNA primer (Leeet al., Nuc. Acids. Res. 20:2471, 1992). This process offers theadvantage of not having to synthesize dye labeled primers. Furthermore,dye-terminator reactions are more convenient in that all four reactionscan be performed in the same tube.

Both dye-primer and dye-terminator methods may be automated using anautomated sequencing instrument produced by Applied Biosystems (FosterCity, Calif.) (U.S. Pat. No. 5,171,534, which is herein incorporated byreference). When using the instrument, the completed sequencing reactionmixture is fractionated on a denaturing polyacrylamide gel orcapillaries mounted in the instrument. A laser at the bottom of theinstrument detects the fluorescent products as they areelectrophoretically separated according to size through the gel.

Two types of fluorescent dyes are commonly used to label the terminatorsused for dye-terminator sequencing-negatively charged and zwitterionicfluorescent dyes. Negatively charged fluorescent dyes include those ofthe fluorescein and BODIPY families. BODIPY dyes(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) are described inInternational Patent Publication WO 97/00967, which is incorporatedherein by reference. Zwitterionic fluorescent dyes include those of therhodamine family. Commercially available cycle sequencing kits useterminators labeled with rhodamine derivatives. However, therhodamine-labeled terminators are rather costly and the product must beseparated from unincorporated dye-ddNTPs before loading on the gel sincethey co-migrate with the sequencing products. Rhodamine dye familyterminators seem to stabilize hairpin structures in GC-rich regions,which causes the products to migrate anomalously. This can involve theuse of dITP, which relaxes the secondary structure but also affects theefficiency of incorporation of terminator.

In contrast, fluorescein-labeled terminators eliminate the separationstep prior to gel loading since they have a greater net negative chargeand migrate faster than the sequencing products. In addition,fluorescein-labeled sequencing products have better electrophoreticmigration than sequencing products labeled with rhodamine. Althoughwild-type Taq DNA polymerase does not efficiently incorporateterminators labeled with fluorescein family dyes, this can now beaccomplished efficiently by use of the modified enzymes as described inU.S. Patent Application Publication No. 2002/0142333, which isincorporated by reference herein in its entirety. Accordingly,modifications as described in US 2002/0142333 can be used in the contextof the present invention to produce fluorescein-family-dye-incorporatingthermostable polymerases having improved primer extension rates. Forexample, in certain embodiments, the unmodified DNA polymerase inaccordance with the present invention is a modified thermostablepolymerase as described in US 2002/0142333 and having the motif setforth in SEQ ID NO:8 (or a motif of SEQ ID NO:9, 10 or 11), andoptionally the motif of SEQ ID NO:27.

Other exemplary nucleic acid sequencing formats in which the mutant DNApolymerases of the invention can be used include those involvingterminator compounds that include 2′-PO₄ analogs of ribonucleotides(see, e.g., U.S. Application Publication Nos. 2005/0037991 and2005/0037398, and U.S. patent application Ser. No. 12/174,488, which areeach incorporated by reference).

Kits

In another aspect of the present invention, kits are provided for use inprimer extension methods described herein. In some embodiments, the kitis compartmentalized for ease of use and contains at least one containerproviding a DNA polymerase of the invention having increased 3′ mismatchdiscrimination in accordance with the present invention. One or moreadditional containers providing additional reagent(s) can also beincluded. Such additional containers can include any reagents or otherelements recognized by the skilled artisan for use in primer extensionprocedures in accordance with the methods described above, includingreagents for use in, e.g., nucleic acid amplification procedures (e.g.,PCR, RT-PCR), DNA sequencing procedures, or DNA labeling procedures. Forexample, in certain embodiments, the kit further includes a containerproviding a 5′ sense primer hybridizable, under primer extensionconditions, to a predetermined polynucleotide template, or a primer paircomprising the 5′ sense primer and a corresponding 3′ antisense primer.In some embodiments, the kit includes one or more containers containingone or more primers that are fully complementary to single nucleotidepolymorphisms or multiple nucleotide polymorphisms, wherein the primersare useful for multiplex reactions, as described above. In other,non-mutually exclusive variations, the kit includes one or morecontainers providing nucleoside triphosphates (conventional and/orunconventional). In specific embodiments, the kit includesalpha-phosphorothioate dNTPs, dUTP, dITP, and/or labeled dNTPs such as,e.g., fluorescein- or cyanin-dye family dNTPs. In still other,non-mutually exclusive embodiments, the kit includes one or morecontainers providing a buffer suitable for a primer extension reaction.In some embodiments, the kit includes one or more labeled or unlabeledprobes. Examples of probes include dual-labeled FRET (fluorescenceresonance energy transfer) probes and molecular beacon probes. Inanother embodiment, the kit contains an aptamer, e.g., for hot start PCRassays.

Reaction Mixtures

In another aspect of the present invention, reaction mixtures areprovided comprising the polymerases with increased 3′-mismatchdiscrimination activity, as described herein. The reaction mixtures canfurther comprise reagents for use in, e.g., nucleic acid amplificationprocedures (e.g., PCR, RT-PCR), DNA sequencing procedures, or DNAlabeling procedures. For example, in certain embodiments, the reactionmixtures comprise a buffer suitable for a primer extension reaction. Thereaction mixtures can also contain a template nucleic acid (DNA and/orRNA), one or more primer or probe polynucleotides, nucleosidetriphosphates (including, e.g., deoxyribonucleotides, ribonucleotides,labeled nucleotides, unconventional nucleotides), salts (e.g., Mn²⁺,Mg²⁺), and labels (e.g., fluorophores). In some embodiments, thereaction mixture further comprises double stranded DNA binding dyes,such as SYBR green, or double stranded DNA intercalating dyes, such asethidium bromide. In some embodiments, the reaction mixtures contain a5′-sense primer hybridizable, under primer extension conditions, to apredetermined polynucleotide template, or a primer pair comprising the5′-sense primer and a corresponding 3′ antisense primer. In certainembodiments, the reaction mixture further comprises a fluorogenic FREThydrolysis probe for detection of amplified template nucleic acids, forexample a Taqman® probe. In some embodiments, the reaction mixturecontains two or more primers that are fully complementary to singlenucleotide polymorphisms or multiple nucleotide polymorphisms. In someembodiments, the reaction mixtures contain alpha-phosphorothioate dNTPs,dUTP, dITP, and/or labeled dNTPs such as, e.g., fluorescein- orcyanin-dye family dNTPs.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Identification of Mutant DNA Polymerases with Increased3′-Mismatch Discrimination

The control DNA polymerase of this example is a Thermus sp. Z05 DNApolymerase of SEQ ID NO:1 except that the amino acid at position 580 isglycine (e.g., a D580G substitution) (hereinafter Z05 D580G polymerase).

Mutations in Z05 D580G polymerase were identified that provide a reducedability to extend an oligonucleotide primer with a 3′-mismatch to atemplate. In brief, the steps in this screening process included librarygeneration, expression and partial purification of the mutant enzymes,screening of the enzymes for the desired property, DNA sequencing,clonal purification, and further characterization of selected candidatemutants. Each of these steps is described further below.

Clonal Library generation: A nucleic acid encoding the polymerase domainof Z05 D580G DNA polymerase was subjected to error-prone (mutagenic) PCRbetween Blp I and Bgl II restriction sites of a plasmid including thisnucleic acid sequence. The amplified sequence is provided as SEQ IDNO:33. The primers used for this are given below:

Forwar Primer:  (SEQ ID NO: 31) 5′-CTACCTCCTGGACCCCTCCAA-3′; and,Reverse Primer: (SEQ ID NO: 32) 5′-ATAACCAACTGGTAGTGGCGTGTAA-3′.PCR was performed using a range of Mg²⁺ concentrations from 1.8-3.6 mM,in order to generate libraries with a range of mutation rates. Bufferconditions were 50 mM Bicine pH 8.2, 115 mM KOAc, 8% w/v glycerol, and0.2 mM each dNTPs. A GeneAmp® AccuRT Hot Start PCR enzyme was used at0.15 U/μL. Starting with 5×10⁵ copies of linearized Z05 D580G plasmidDNA per reaction volume of 50 μL, reactions were denatured using atemperature of 94° C. for 60 seconds, then 30 cycles of amplificationwere performed, using a denaturation temperature of 94° C. for 15seconds, an annealing temperature of 60° C. for 15 seconds, an extensiontemperature of 72° C. for 120 seconds, and followed by a final extensionat a temperature of 72° C. for 5 minutes.

The resulting amplicon was purified with a QIAquick PCR Purification Kit(Qiagen, Inc., Valencia, Calif., USA) and cut with Blp I and Bgl II, andthen re-purified with a QIAquick PCR Purification Kit. A Z05 D580Gvector plasmid was prepared by cutting with the same two restrictionenzymes and treating with alkaline phosphatase, recombinant (RAS, cat#03359123001) and purified with a QIAquick PCR Purification Kit. The cutvector and the mutated insert were mixed at a 1:3 ratio and treated withT4 DNA ligase for 5 minutes at room temperature (NEB Quick Ligation™Kit). The ligations were purified with a QIAquick PCR Purification Kitand transformed into an E. coli host strain by electroporation.

Aliquots of the expressed cultures were plated on ampicillin-selectivemedium in order to determine the number of unique transformants in eachtransformation. Transformations were stored at −70° C. to −80° C. in thepresence of glycerol as a cryo-protectant.

Each library was then spread on large format ampicillin-selective agarplates. Individual colonies were transferred to 384-well platescontaining 2× Luria broth with ampicillin and 10% w/v glycerol using anautomated colony picker (QPix2, Genetix Ltd). These plates wereincubated overnight at 30° C. to allow the cultures to grow and thenstored at −70° C. to −80° C. The glycerol added to the 2× Luria brothwas low enough to permit culture growth and yet high enough to providecryo-protection. Several thousand colonies at several mutagenesis (Mg²⁺)levels were prepared in this way for later use.

Extract library preparation Part 1—Fermentation: From the clonallibraries described above, a corresponding library of partially purifiedextracts suitable for screening purposes was prepared. The first step ofthis process was to make small-scale expression cultures of each clone.These cultures were grown in 96-well format; therefore there were 4expression culture plates for each 384-well library plate. 0.5 μL wastransferred from each well of the clonal library plate to a well of a 96well seed plate, containing 150 μl, of Medium A (see Table 3 below).This seed plate was shaken overnight at 1150 rpm at 30° C., in an iEMSplate incubator/shaker (ThermoElectron). These seed cultures were thenused to inoculate the same medium, this time inoculating 20 μL into 250μL Medium A in large format 96 well plates (Nunc # 267334). These plateswere incubated overnight at 37° C. with shaking. The expression plasmidcontained transcriptional control elements, which allow for expressionat 37° C. but not at 30° C. After overnight incubation, the culturesexpressed the clone protein at typically 1-10% of total cell protein.The cells from these cultures were harvested by centrifugation. Thesecells were either frozen (−20° C.) or processed immediately, asdescribed below.

TABLE 2 Medium A (Filter-sterilized prior to use) ComponentConcentration MgSO₄•7H₂O 0.2 g/L Citric acid•H₂O 2 g/L K₂HPO₄ 10 g/LNaNH₄PO₄•4H₂O 3.5 g/L MgSO₄ 2 mM Casamino acids 2.5 g/L Glucose 2 g/LThiamine-HCl 10 mg/L Ampicillin 100 mg/L

Extract library preparation Part 2—Extraction: Cell pellets from thefermentation step were resuspended in 25 μL Lysis buffer (Table 3 below)and transferred to 384-well thermocycler plates and sealed. Note thatthe buffer contained lysozyme to assist in cell lysis, and DNase toremove DNA from the extract. To lyse the cells the plates were incubatedat 37° C. for 15 minutes, frozen overnight at −20° C., and incubatedagain at 37° C. for 15 minutes. Ammonium sulfate was added (1.5 μL of a2 M solution) and the plates incubated at 75° C. for 15 minutes in orderto precipitate and inactivate contaminating proteins, including theexogenously added nucleases. The plates were centrifuged at 3000×g for15 minutes at 4° C. and the supernatants transferred to a fresh 384-wellthermocycler plate. These extract plates were frozen at −20° C. forlater use in screens. Each well contained about 0.5-3 μM of the mutantlibrary polymerase enzyme.

TABLE 3 Lysis Buffer Component Concentration or Percentage Tris pH 7.550 mM EDTA 1 mM MgCl₂ 6 mM Tween 20 0.5% v/v Lysozyme (from powder) 1mg/mL DNase I 0.05 Units/μL

Screening extract libraries for reduced 3′ primer mismatch extensionrate: The extract library was screened by comparing the extension rateof a primer perfectly matched to an oligonucleotide template vs. theextension rate of a primer with a 3′ G:T mismatch.

The enzyme extracts above were diluted 10-fold for primer extensionreactions by combining 2.5 μl extract with 22.5 μl of a buffercontaining 20 mM Tris-HCl, pH 8, 100 mM KCl, 0.1 mM EDTA, and 0.2%Tween-20 in a 384-well thermocycler plate, covering and heating for 10minutes at 90° C. Control reactions with perfect match primer combined0.5 μl of the diluted extract with 15 μl master mix in 384-well PCRplates. Extension of the primed template was monitored every 10 secondsin a modified kinetic thermal cycler using a CCD camera (see, Watson,supra). Master mix contained 50 nM primed primer template, 25 mMTricine, pH 8.3, 100 mM KOAc, 0.6×SYBR Green 1,200 μM each dNTP, 100 nMAptamer, and 2.5 mM Magnesium Acetate. In order to distinguishextension-derived fluorescence from background fluorescence, parallelwells were included in the experiment in which primer strand extensionwas prevented by leaving out the nucleotides from the reaction mastermix. Reactions with the 3′-mismatched primer were performed as aboveexcept 1.5 ul the diluted extract was added to each reaction and 1.5 mMManganese Acetate was substituted for the Magnesium Acetate. Increasingthe amount of extract three fold and using Manganese as the metalactivator both make mismatch extension more likely and therefore improvethe selectivity of the screen for those enzymes with the greatestability to discriminate against 3′-mismatch extension.

Approximately 5000 mutant extracts were screened using the aboveprotocol. Approximately 7% of the original pool was chosen forrescreening based on a perfect match primer extension value above anarbitrary cutoff and low mismatch to perfect match extension ratio.Culture wells corresponding to the top extracts were sampled to freshgrowth medium and re-grown to produce a new, culture plates containingthe best mutants, as well as a number of parental cultures to be usedfor comparison. These culture plates were then used to make freshextracts which were rescreened to confirm the original screen phenotype.The primer extension rates for the reactions with the perfect 3′-matchedand the 3′-mismatched primers were calculated as the slope of the risein fluorescence over time for the linear portion of the curve. The ratioof mismatched extension slope divided by the perfect matched extensionslope was used to rank and select the best candidates. Selected clonesfrom the rescreening, plus for comparison the parental clone Z05 D580G,with their respective genotypes and phenotypes are included in the tablebelow.

TABLE 4 Perfect Match Mismatch MM Slope/ Enzyme Slope Slope PM Slope Z05D580G 8.29 8.04 0.97 Z05 D580G Y547C 11.76 0.84 0.07 Z05 D580G E475D12.43 1.58 0.13 Y547N R632S

Example 2 Amplification of Mutant BRAF Plasmid Template in a Backgroundof Wild-Type BRAF Human Genomic Template

The control DNA polymerase of this example is a Thermus sp. Z05 DNApolymerase of SEQ ID NO:1 except that the amino acid at position 580 isGlycine (e.g., a D580G substitution) (hereinafter ZO5 D580G polymerase).

Purified ZO5 D580G Y547C and ZO5 D580G E475D Y547NR632S were compared tothe parental enzyme Z05 D580G in TaqMan PCR for improved discriminationof a mutant BRAF V600R target in a background of wild-type Human GenomicDNA.

Primers were used that amplify a region of the human BRAF gene and areperfectly matched to the target when said target carries a mutation incodon 600 of BRAF, V600K. Against wild-type BRAF target, present inhuman genomic DNA, the allele selective primer results in a single A:Cmismatch at the 3′ end. The common primer is perfectly matched to theBRAF gene, as is the probe sequence, which allows for real-time, TaqMandetection of amplification. Each reaction had 10,000 copies (33 ng) ofwild-type Human Genomic cell line DNA, or either 10,000 or 100 copies ofa linearized plasmid containing the BRAF V600R mutant sequence in afinal volume of 16 μl. To allow for the different salt optima of theenzymes, amplifications were performed using a range of KClconcentrations from 25 to 130 mM. Buffer conditions were 50 mM Tris-HClpH 8.0, 2.5 mM MgCl₂, 0.2 mM each dNTP, 0.02 U/μl UNG, and 200 nMAptamer. Forward and Reverse primers were at 100 nM and the probe was at25 nM. All DNA polymerases were assayed at 20 nM and add 2% (v/v) enzymestorage buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mMEDTA, 1 mM DTT, 0.5% Tween 20) to the reactions. The reactions wereperformed in a Roche LightCycler 480 thermal cycler and denatured usinga temperature of 95° C. for 60 seconds, then 99 cycles of amplificationwere performed, using a denaturation temperature of 92° C. for 10seconds and an annealing temperature of 62° C. for 30 seconds.

Reactions were run in duplicate, crossing points (“Cps”) were calculatedby the Abs Quant/2^(nd) derivative Max method and the Cps were averaged.The averaged Cp values are shown in the table below as well ascalculated PCR efficiency and discrimination factor values at the KClconcentration for each enzyme which resulted in the earliest high copymutant Cp. High Copy delta Cp is equal to the difference between theaverage Cp values of the reactions with 10,000 copy of 3′-mismatchedwild-type genomic target and the average Cp values of the reactions with10,000 copy of perfect match plasmid target in a background of 10,000copy of 3′-mismatched wild-type genomic target. All reactions have abackground of 10,000 copy wild type BRAF target, therefore the Cps ofthe reactions with no mutant plasmid represent breakthroughamplification of the mismatched primer template and the limit ofdiscrimination for that enzyme under the condition tested. Z05 D580GY547C and Z05 D580G E475D Y547N R632S showed better discrimination thanthe parental Z05 D580G.

TABLE 5 Cps of Amplification of BRAF V600K mutant plasmid vs. Humangenomic DNA 1000 10,0000 Optimum 0 copies copies copies High KCL mutantmutant mutant % PCR Discrimination copy Enzyme (mM) plasmid plasmidplasmid Efficiency Factor ΔCp Z05 D580G 120 34.0 32.2 26.1 110 2.6 8 Z05D580G Y547C 100 52.8 34.9 27.7 90 7.0 25 Z05 D580G E475D 40 46.8 34.928.4 104 5.7 18 Y547N R632S

This example demonstrates that the Y547C and Y547N mutant enzymes haveimproved rare allele detection relative to the control parental enzyme,Z05 D580G.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, sequence accessionnumbers, patents, and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes.

1. A DNA polymerase having increased 3′-mismatch discrimination activitycompared with a control DNA polymerase, wherein the amino acid of theDNA polymerase corresponding to position 547 of SEQ ID NO:1 is any aminoacid other than Y, and wherein the control DNA polymerase has the sameamino acid sequence as the DNA polymerase except that the amino acid ofthe control DNA polymerase corresponding to position 547 of SEQ ID NO:1is Y.
 2. The DNA polymerase of claim 1, wherein the amino acid of theDNA polymerase corresponding to position 547 of SEQ ID NO:1 is selectedfrom G, A, L, M, W, P, S, T, F, C, N, Q, D, E, K, R, V, I or H.
 3. TheDNA polymerase of claim 1, comprising a motif in the polymerase domaincomprising K-L-X₁-X₂-T-X₃-X₄-X₅-X₆-X₇-X₈, wherein: X₁ is K, R or Q; X₂is N, S, or G; X₃ is an amino acid other than Y; X₄ is V, I or L; X₅ isD or E; X₆ is P, A, T, S or G; X₇ is L or I; and X₈ is P or L (SEQ IDNO:8).
 4. The DNA polymerase of claim 3, wherein X₃ is selected from G,A, L, M, W, P, S, T, F, C, N, Q, D, E, K, R, V, I or H (SEQ ID NO:42).5. The DNA polymerase of claim 1, comprising a motif in the polymerasedomain comprising K-L-K-X₂-T-X₃-X₄-D-X₆-L-P, wherein: X₂ is N or S; X₃is an amino acid other than Y; X₄ is V or I; and X₆ is P or A (SEQ IDNO:9).
 6. The DNA polymerase of claim 1, comprising a motif in thepolymerase domain comprising K-L-K-X₂-T-X₃-X₄-D-P-L-P, wherein: X₂ is Sor N; X₃ is an amino acid other than Y; and X₄ is V or I (SEQ ID NO:10).7. The DNA polymerase of claim 6, wherein X₃ is C or N (SEQ ID NO:11).8. The DNA polymerase of claim 1, wherein the amino acid correspondingto position 580 of SEQ ID NO:1 is any amino acid other than D or E. 9.The DNA polymerase of claim 1, wherein the amino acid corresponding toposition 580 of SEQ ID NO:1 is selected from the group consisting of L,G, T, Q, A, S, N, R, and K.
 10. The DNA polymerase of claim 6, whereinthe DNA polymerase comprises an amino acid sequence at least 80%sequence identical to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 36, 37, 38, 39, 40or
 41. 11. The DNA polymerase of claim 6, wherein the polymerase has atleast 80% sequence identity to SEQ ID NO:1.
 12. The DNA polymerase ofclaim 11, wherein the DNA polymerase is a Z05 DNA polymerase, and theamino acid at position 580 is selected from the group consisting of L,G, T, Q, A, S, N, R, and K.
 13. A recombinant nucleic acid encoding theDNA polymerase according to claim
 1. 14. A method for conducting primerextension, comprising: contacting a DNA polymerase according to claim 1with a primer, a polynucleotide template, and nucleoside triphosphatesunder conditions suitable for extension of the primer, thereby producingan extended primer.
 15. The method of claim 14, wherein the primerextension method is a method for conducting polymerase chain reaction(PCR).
 16. A kit for producing an extended primer, comprising: at leastone container providing a DNA polymerase according to claim
 1. 17. Thekit according to claim 16, further comprising one or more additionalcontainers selected from the group consisting of: (a) a containerproviding a primer hybridizable, under primer extension conditions, to apredetermined polynucleotide template; (b) a container providingnucleoside triphosphates; and (c) a container providing a buffersuitable for primer extension.
 18. A reaction mixture comprising a DNApolymerase according to claim 1, at least one primer, a polynucleotidetemplate, and nucleoside triphosphates.